Vacuolar protein sorting ortholog 35 (VPS35) is a protein involved in autophagy and is implicated in neurodegenerative diseases, such as Parkinson's disease (PD) and Alzheimer's disease (AD). VPS35 is part of a complex called the retromer, which is responsible for transporting select cargo proteins between vesicular structures (e.g., endosomes, lysosomes, vacuoles) and the Golgi apparatus. Mutations in the VPS35 gene (VPS35) cause aberrant autophagy, where cargo proteins fail to be transported and dysfunctional or unnecessary proteins fail to be degraded. There are numerous pathways affected by altered VPS35 levels and activity, which have clinical significance in neurodegeneration. There is therapeutic relevance for VPS35, as interventions aimed at correcting VPS35 function are in speculation.
Gene In humans, VPS35 is transcribed on chromosome 16q11.2 where is spans about 29.6 kilobases and contains 17 exons. It is evolutionarily conserved and required for survival, as mouse knockout studies have demonstrated embryonic lethality. VPS35 levels peak at postnatal days 10-15 and then decline to a low, stable level throughout adulthood. RNA expression of VPS35 is ubiquitous throughout the body, but are higher in the brain, heart, gonads, spleen, and skeletal muscle, and lower in the lung, liver, kidney, and blood leukocytes.
Protein VPS35 was first identified in Saccharomyces cerevisiae from a study investigating the formation of lysosome-like vacuoles and sorting of vacuolar proteins. The protein contains 796 amino acid residues, with a molecular mass of 92 kDa and an isoelectric point of 5.32.
Structure VPS35 binds with other proteins to form the retromer, an evolutionarily conserved complex that plays a major role in transmembrane protein recycling from endosomes to the trans-Golgi network (TGN). VPS35 itself folds into a secondary structure that represents an α-helical solenoid, containing 34 α-helix repeats.
As part of the retromer, VPS35 trimerizes with other vacuolar protein sorting orthologs, VPS26 and VPS29. In less common situations, VPS35 can bind VPS26 and VPS29 alone, creating heterodimers. VPS26 binds the N-terminus of VPS35 at a conserved PRLYL motif (residues 1-172), whereas a C-terminal α-solenoid fold (residues 307-796) binds VPS29. These VPS orthologs stabilize each other within the retromer; VPS35 knockdown can lead to VPS29 degradation, and vice versa. The VPS35, VPS26, and VPS29 trimer forms the cargo recognition complex, a necessary component for the retromer's ability to regulate vesicular sorting. This is achieved by specifically targeting sorting nexins (i.e., SNX1, SNX2, SNX5, SNX6, and SNX32), which anchor the retromer to endosomes and other vesicular structures.
Function
After translation, VPS35 localizes to the endosome, where it, along with the retromer, has been studied in various phagocytotic processes. Sortilin and the cation-independent mannose-6-phosphate receptor (CI-MPR) regulate lysosome-mediated degradation and are known VPS35 cargo-proteins. The Wiskott Aldrich Syndrome protein and scar homologue (WASH) complex, directly recruited by VPS35, regulates macroautophagy, whereby an autophagosome engulfs select proteins and whole organelles for lysosomal degradation. VPS35 also recruits lysosome-associated membrane protein 2a (LAMP2a) to help initiate chaperone-mediated autophagy, whereby a heat shock protein 70kDa protein 8 (HSPA8)-containing complex facilitates degradation of proteins containing a KFERQ signal.
Mutations in VPS35 lead to generalized endosomal defects, such as vesicle enlargement and sequestration around the nucleus. These mutations also affect many cellular processes overseen by the retromer complex, including vesicular trafficking, plasma membrane receptor recycling, protein aggregation, mitochondrial function, and dopamine signaling. These processes play a role in neurodegenerative diseases.
Clinical significance
Parkinson's disease PD is a clinical challenge, presenting itself as the second most common neurodegenerative disease. It is characterized by dopaminergic neuron loss in the substantia nigra pars compacta and the toxic aggregation of the α-synuclein protein into Lewy Bodies, all of which cause motor dysfunctions and dopamine deficiency in PD patients. VPS35 mutations and dysfunctional retromer-mediated protein sorting and recycling are implicated in neurodegenerative processes relevant to PD.
Genetic variants In 2011, exome sequencing was performed on a Swiss family where all six members developed late-onset, autosomal dominant PD. The sequencing revealed a mutation from an aspartic acid to asparagine at amino acid residue 620 (D620N) in VPS35. Sequence alignment of VPS35 orthologs between Homo sapiens, Pan troglodytes, Mus musculus, Rattus norvegicus, Bos taurus, Canis familiaris, Gallus gallus, Xenopus laevis, Danio rerio, Drosophila melanogaster, and Saccharomyces cerevisiae has shown that the 620 position is highly mutagenic with a propensity to substitute aspartic acid with asparagine throughout evolution. Geographically, the VPS35-D620N variant is most prevalent among Caucasian descendants and families in Switzerland, Austria, the United States, Tunisia, Israel, the United Kingdom, France, Germany, and Japan. Other VPS35 variants (e.g., P316S, R32S, R524W, I560T, H599R, M607V) have been identified among some PD patients; however, the VPS35-D620N variant has been the most extensively characterized and is currently the only mutated form of VPS35 confirmed to be pathogenic. It is currently unknown how frequently the D620N mutation appears in the general population.
Patient characteristics The neurochemistry and clinical presentation between VPS35-induced PD and idiopathic PD are not significantly different. PD patients with VPS35 mutations experience typical PD symptoms: bradykinesia (91.4%), rigidity (80%), tremor (77.1%), and postural instability (60%). Patients with the VPS35-D620N variant experience a mean age of disease onset between 50–52 years (earlier than classic PD onset of 65–85 years), are typically white (82.9%), and have high familial history (91.4%). The VPS35-D620N mutation occurs more frequently in familial PD (~1.3%) than in sporadic PD (0.3%). PD patients harboring the VPS35-D620N mutation follow slow disease progression; cognitive or neuropsychiatric aspects are generally spared. All patients respond well to levodopa therapy, one of the main treatments to alleviate PD symptoms. Overall, the VPS35-D620N mutation is relatively rare in PD, with estimated prevalence of 0.115% from over a dozen case studies comprising approximately 22,000 PD patients globally. One clinical caveat is that Lewy body pathology has not been fully understood in the context of VPS35-induced PD, as only a single PD patient harboring the VPS35-D620N variant has undergone an autopsy. However, it is known that in brains of sporadic PD patients, there is mislocalized VPS35 in Lewy bodies.
Interactions between VPS35 and PD-linked genes Changes in VPS35 affect levels of leucine-rich repeat kinase 2 (LRRK2), a candidate gene involved in PD that helps vesicular trafficking by phosphorylating Rab proteins. VPS35 knockdown or overexpression of the VPS35-D620N variant can enhance autophosphorylation of LRRK2, increasing its overall activity. Transgenic mice harboring the VPS35-D620N variant have demonstrated altered Rab phosphorylation by LRRK2. In Drosophila, overexpression of VPS35 can rescue mutant LRRK2-induced PD phenotypes. The LRRK2 mutation, G2019S, has also been found to decrease VPS35 levels in mouse N2A neuroblastoma cells, indicating an affective mechanism between these factors.
Parkin, an E3 ubiquitin ligase involved in protein degradation, is commonly seen in autosomal recessive juvenile parkinsonism and has known interactions with VPS35. VPS35 overexpression in Drosophila lacking Parkin reverses Parkin-deficient phenotypes, increasing longevity, climbing ability, and decreasing sensitivity to paraquat, a toxic herbicide known to be associated with PD onset in humans. Parkin is known to increase ubiquitination of VPS35. Counterintuitively, this does not lead to proteasomal degradation of VPS35; a functional role is still being investigated. It is proposed that VPS35 may act downstream of Parkin, since overexpression of VPS35-D620N in Parkin knockout mice has not been shown to rescue dopaminergic neuron loss.
Alzheimer's disease AD is the most prominent cause of dementia (60-80%) and affects many cognitive abilities in patients, including word retrieval, memory recall, and other general executive functions necessary for basic self-care. AD pathology typically begins with the formation of amyloid beta (Aβ) plaques in the brain, which trigger an inflammatory response by microglia and cause a cascade of tau accumulation and spreading. These changes result in the degeneration of neurons, leading to a loss of synaptic connections and neurotransmitter signaling.
Clinically, low expression of VPS35 in the brain is a risk factor of AD, since it is known that regions high in AD pathology show low VPS35 activity. Specifically, there are decreased levels of VPS35 in the hippocampi of postmortem AD patients relative to healthy patients. This can be modeled in a specific strain of AD-like mice, Tg2576, whereby heterozygous deletion of VPS35 enhances AD phenotypes in the hippocampus and cortex. Humans possessing a VPS35 genetic variant also increases the risk of developing AD. Consequently, AD pathology is linked to aberrant retromer function.
VPS35 knockdown studies have demonstrated increased amyloid precursor protein (APP) and Aβ plaque levels, hallmarks of AD. VPS35 insufficiency reduces transport of endosomes containing APP, ultimately facilitating APP aggregation and formation of Aβ plaques. Sortilin-related receptor is a cargo protein that interacts with VPS35; it binds APP and delivers APP to the lysosomal system for degradation. Impairment of retromer activity by VPS35 mutation also increases beta-secretase 1 (BACE1) activity, which cleaves larger APPs and enhances Aβ plaque toxicity. This is observed in heterozygous mice deficient in VPS35, which have greater amounts of Aβ40 and Aβ42 compared to controls. Decreased expression of VPS35 in Drosophila AD models further shows an increase in Aβ plaque formation, BACE1 activity, memory deficits, and synaptic dysfunction.
VPS35 deletion in mammalian models of AD is associated with aberrant microglia function and impaired hippocampal development; however, causal variants have yet to be determined. Possibly, VPS35 loss-of-function may impair an inflammatory response in AD, since triggering receptor expressed on myeloid cells 2 (TREM2), a microglial factor associated with inflammation in AD, is a cargo protein of the retromer and VPS35. Tau pathology is also observed in the brains of VPS35-D620N knock-in mice.
Pathophysiology
Aberrant autophagy