Proteomics Analysis of a Human Brain Sample from a Mucolipidosis Type IV

Vardi et al. Orphanet J Rare Dis (2021) 16:39 https://doi.org/10.1186/s13023-021-01679-7 RESEARCH Open Access Proteomics analysis of a human brain sample from a mucolipidosis type IV patient reveals pathophysiological pathways Ayelet Vardi1, Amir Pri‑Or2, Noa Wigoda3, Yulia Grishchuk4 and Anthony H. Futerman1* Abstract Background: Mucolipidosis type IV (MLIV), an ultra‑rare neurodevelopmental and neurodegenerative disorder, is caused by mutations in the MCOLN1 gene, which encodes the late endosomal/lysosomal transient receptor potential channel TRPML1 (mucolipin 1). The precise pathophysiogical pathways that cause neurological disease in MLIV are poorly understood. Recently, the frst post‑mortem brain sample became available from a single MLIV patient, and in the current study we performed mass spectrometry (MS)‑based proteomics on this tissue with a view to delineat‑ ing pathological pathways, and to compare with previously‑published data on MLIV, including studies using the / Mcoln1− − mouse. Results: A number of pathways were altered in two brain regions from the MLIV patient, including those related to the lysosome, lipid metabolism, myelination, cellular trafcking and autophagy, mTOR and calmodulin, the comple‑ ment system and interferon signaling. Of these, levels of some proteins not known previously to be associated with MLIV were altered, including APOD, PLIN4, ATG and proteins related to interferon signaling. Moreover, when proteins / detected by proteomics in the human brain were compared with their orthologs detected in the Mcoln1− − mouse by RNAseq, the results were remarkably similar. Finally, analysis of proteins in human and mouse CSF suggest that calbindin 1 and calbindin 2 might be useful as biomarkers to help chart the course of disease development. Conclusions: Despite the sample size limitations, our fndings are consistent with the relatively general changes in lysosomal function previously reported in MLIV, and shed light on new pathways of disease pathophysiology, which is required in order to understand the course of disease development and to determine the efcacy of therapies when they become available for this devastating disease. Keywords: Lysosome, TRPML1, Brain, Neuropathology, Autophagy, Neuroinfammation, Calbindin Introduction TRPML1 (mucolipin 1) [2]. Over two dozen mutations Mucolipidosis type IV (MLIV), an ultra-rare lysoso- in MCOLN1 have been identifed which lead to MLIV mal storage disease (LSD) primarily found in the Ash- [3]. TRPML1 is a non-selective cation channel that regu- kenazi Jewish population [1], is caused by mutations lates lysosomal ion content [4, 5]. TRPML1 also plays a in the MCOLN1 gene, which encodes the late endo- role in autophagy regulation, intracellular trafcking and somal/lysosomal transient receptor potential channel in the mechanistic target of rapamycin (mTOR)/tran- scription factor EB (TFEB) signaling axis [6, 7]. MLIV is a neurodevelopmental and neurodegenerative disorder *Correspondence: [email protected] 1 Department of Biomolecular Sciences, Weizmann Institute of Science, with severe psychomotor developmental delay, impaired 76100 Rehovot, Israel gastric function, mental retardation, motor defcits and Full list of author information is available at the end of the article progressive visual impairment [1, 8]. At present there © The Author(s) 2021. 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The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Vardi et al. Orphanet J Rare Dis (2021) 16:39 Page 2 of 13 is no therapy for MLIV and our understanding of brain appended with common laboratory protein contami- pathophysiology is limited due to the lack of human tis- nants. Quantitative comparisons were calculated using sue samples available for research. Perseus v1.6.0.7. Pathway analysis was performed using We recently obtained brain tissue and cerebrospi- Gene Analytics [14]. Proteins were categorized based nal fuid (CSF) from a 36-year-old male who died from on the Human Protein Atlas [15] (http://www.prote inatl MLIV-related complications. Tis is the frst time that as.org). post-mortem brain tissue has been made available from a MLIV patient. We performed mass spectrometry (MS)- CSF MS‑based proteomics based proteomics on this tissue with a view to delineat- Samples were lysed with 8 M urea and incubated at room ing pathological pathways. Moreover, we compared our temperature for 30 min prior to incubation with 5 mM data with that obtained using a mouse model of MLIV, dithiothreitol (Sigma-Aldrich) at room temperature for namely the Mcoln1−/− mouse [9, 10], which further 1 h, followed by alkylation with 10 mM iodoacetamide validated that the Mcoln1−/− mouse is a genuine animal (Sigma-Aldrich) in the dark at room temperature for model of MLIV, and of more importance, allowed us to 45 min. Samples were diluted in 2 M urea with 50 mM identify some novel pathological components and path- ammonium bicarbonate. Proteins were digested with ways in the human MLIV brain tissue, such as activation trypsin (50:1; w/vol) (Promega, Madison, WI, USA) over- of complement and interferon (IFN) pathways and lipid night at 37 °C followed by a second trypsin digestion for droplet and lipoprotein metabolism. Along with the iden- 4 h. Digestion was terminated using trifuoracetic acid tifcation of a potential biomarker (calbindin 2) for early (1% fnal concentration). Peptides were desalted using disease development in the CSF, our study paves the way an Oasis hydrophilic lipophilic balance column (Waters) for further understanding of the molecular pathways that and then vacuum dried and stored at -80 °C. Te result- lead to disease pathophysiology in MLIV, a pre-requisite ing peptides were analyzed using nanofow liquid chro- for development of putative therapies. matography (nanoACQUITY, Waters) coupled to high resolution, high mass accuracy mass spectrometry Methods (Q Exactive HFX for mouse CSF or Fusion Lumos for Human brain and CSF samples human CSF, Termo Scientifc). Samples were analyzed Human brain and CSF samples were obtained from the in random order in discovery mode. Te raw data was NIH NeuroBioBank Brain and Tissue Repository at the processed with MaxQuant v1.6.0.16 [13] and analyzed University of Maryland, Baltimore. Te post mortem with the Andromeda search engine against the mouse MLIV patient was a 36 year-old male and the control or human proteome database (www.unipr ot.com), and was a 34-year-old healthy male who died from unrelated appended with common laboratory protein contami- causes. nants. Quantitative comparisons were calculated using Perseus v1.6.0.7. Pathway analysis was done using Gene Brain tissue MS‑based proteomics Analytics [14]. Brain tissue was lysed using a GentleMACS dissocia- tor (Miltenyi Biotec, Bergisch Gladbach, Germany) in Mice 50 mM Tris HCl containing 5% sodium dodecyl sulfate Mcoln1−/− mice on a C57Bl/6 J background [10] were (SDS) and supplemented with a protease inhibitor cock- used. Littermates (Mcoln1+/+ or Mcoln1±) were used tail (1:200, Sigma-Aldrich, Missouri, USA). Homogen- as controls and both females and males were used in ates were incubated with 5 mM dithiothreitol for 1 h at all studies. Genotyping was performed by PCR using 56 °C followed by 10 mM iodoacetamide in the dark at genomic DNA extracted from mouse tails. Mice were room temperature for 45 min. Samples were loaded onto maintained in the Experimental Animal Center of the S-Trap microcolumns (Protif, New-York, USA) and Weizmann Institute of Science. Animal experiments subjected to tryptic digestion [11, 12]. Samples were were approved by the Weizmann Institute Institutional vacuum dried and stored at -80 °C. Te resulting pep- Animal Care and Use Committee. tides were analyzed by nanofow liquid chromatography (NanoAcquity, Waters, Milford, MA, USA) coupled to high resolution, high mass accuracy mass spectrometry Mouse CSF (Fusion Lumos, Termo Scientifc, Waltham, MA, USA) 1-, 2-, 3- and 7-month-old Mcoln± or Mcoln1−/− mice and sequentially analyzed in discovery mode. Raw data were anesthetized using 200 mg/ml sodium pentobarbi- was processed with MaxQuant v1.6.0.16 [13]. Data was tal and CSF aspirated from the cisterna magna using a analyzed with the Andromeda search engine against glass needle. CSF samples were kept at -80 °C. the human proteome database (www.unipr ot.com) and Vardi et al. Orphanet J Rare Dis (2021) 16:39 Page 3 of 13 RNAseq Table 1 Lysosomal proteins in human brain and CSF Brain tissue was homogenized using a GentleMACS dis- Protein CB CRB CSF sociator and mRNA was isolated using the RNeasy mini kit (Qiagen GmbH, Hilden, Germany). RNA concentra- Ratio (MLIV/Con) tion (ratio 260/230 and 260/280 nm) was measured using Lysosomal hydrolases a NanoDrop ND-1000 (Termo Scientifc, Waltham, HEXA 1.77 2.93 0.90 MA, USA) and RNA integrity evaluated using an RNA HEXB 5.96 3.95 6.96 NPC1 3.93 n.d screen tape on a Tapestation 2200 (Agilent, Santa Clara, − California, USA). A bulk variation of MARSseq [16] NPC2 2.58 4.34 2.59 GNS 2.94 3.01 was used to construct RNAseq libraries.

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