WO2019235923A1 - Role for low density lipoprotein receptor-related protein 10 in progressive brain diseases - Google Patents

Abstract

The invention relates to methods typing a human subject as suffering from, or being at risk of suffering from, a progressive brain disease. In addition, the invention relates to methods of screening for a level of activity of a low density lipoprotein receptor-related protein, and to methods of screening for a compound that modulates the activity of the low density lipoprotein receptor-related protein. The invention further relates to methods of treating a human subject suffering from, or being at risk of suffering from, a progressive brain disease, comprising providing a stimulator of the low density lipoprotein receptor-related protein, and to a composition comprising a stimulator of the low density lipoprotein receptor-related protein and a pharmaceutically acceptable excipient.

Description

Title: Role for low density lipoprotein receptor-related protein 10 in progressive brain diseases

FIELD

The invention is in the field of diagnostic and medical treatment methods and therapeutic compositions for use in such methods. In particular, the invention relates to therapeutic compounds for treatment of progressive brain diseases, especially Parkinson Disease (PD), Parkinson Disease Dementia (PDD) and Dementia with Lewy bodies (DLB).

INTRODUCTION

Parkinson’s disease (PD), the most common neurodegenerative movement disorder, is clinically defined by hradykinesia, resting tremor, muscular rigidity, and favorable response to levodopa or dopamine -agonist treatment (Obeso et al., 2017. Mov Disord 32: 1264-310). PD pathological hallmarks are loss of nigrostriatal dopaminergic neurons with intracellular inclusions containing alpha-synuclein protein (Lewy bodies, [LBs]; Lewy neurites, [LNs]) in surviving neurons (Obeso et al., 2017. Ibid). Non-motor manifestations, such as olfactory, cognitive, psychiatric, sleep, and autonomic disturbances, are nowadays recognized as frequent and relevant PD features (Obeso et al., 2017. Ibid). Cognitive decline progresses into overt dementia in up to 80% of PD patients (Hely et al., 2008. Mov Disord 23: 837- 442), leading to a diagnosis of Parkinson’s disease dementia (PDD) (McKeith et al., 2005. Neurology 65: 1863-72; McKeith et al., 2017. Neurology 89: 88-100).

Furthermore, in patients with dementia with Lewy bodies (DLB), severe cognitive disturbances are the initial manifestation, often but not always followed by parkinsonism (McKeith et al., 2005. Ibid; McKeith et al., 2017. ibid). DLB accounts for ~5% of dementia cases in the elderly (Hogan et al., 2016. Can J Neurol Sci 43: S83-95) and is associated with severe, widespread brain LB-pathology (McKeith et al., 2017.ibid; Walker et al., 2015. Lancet 386: 1683-97).

Rare, highly-penetrant variants in SNCA (Polymeropoulos et al., 1997.

Science 276: 2045-7; Singleton et al., 2013. Mov Disord 28: 14-23) and LRRK2 (Singleton et al., 2013. Ibid; Zimprich et al., 2004. Neuron 44: 601-7; Paisan-Ruiz et al., 2004. Neuron 44: 595-600) cause hereditary forms of dominantly transmitted, LB-positive PD, PDD, and DLB. Moreover, common variants in SNCA, LRRK2, and GBA, are risk factors for the same disorders (Sidransky et al., 2009. N Engl J Med 361: 1651-61; Nalls et al., 2013. JAMA Neurol 70: 727-35; Chang et al., 2017. Nat Genet 49: 1511-6). Pathological alpha-synuclein misfolding and aggregation appears central in several neurodegenerative diseases including PD and DLB, collectively termed alpha-synucleinop athies (Goedert et al., 2013. Nat Rev Neurol 9: 13-24). Thus, there are clinical, pathological, and molecular overlaps suggesting that PD, PDD and DLB are parts of a continuum of Lewy Body diseases (Langston et al., 2015. Nat Genet 47: 1378-84; Friedman, 2018. Parkinsonism Relat Disord 46: S6-S9). Yet, in most patients with familial forms of PD, PDD or DLB, variants in the above-mentioned genes are not found, suggesting that other causative or predisposing genes remain to be identified.

There is thus a need to identify novel genes implicated in the development of familial PD, PDD and DLB.

SUMMARY OF THE INVENTION

The invention provides a method of typing a human subject as suffering from, being at risk of suffering from, a progressive brain disease, said method comprising a) providing a sample comprising cells, or parts thereof, of said subject; b) determining in said cells or cell parts an amount and/or a level of activity of low density lipoprotein receptor-related protein 10 (LRP10); c) correlating said determined amount and/or level of activity to a healthy control; and d) typing the human subject as suffering from, or being at risk of suffering from, a progressive brain disease if the determined amount and/or level of activity of LRP10 is reduced, when compared to the healthy control.

The present invention is based on the identification of LRP10 gene defects implicated in the development of inherited forms of synucleinopathies such as Parkinson’s Disease. Elucidating the function of the LRP10 protein and pathways can offer novel insights into mechanisms, biomarkers and therapeutic targets.

Said progressive brain disease preferably is selected from Parkinson Disease (PD), Parkinson Disease Dementia (PDD) and Dementia with Lewy bodies (DLB). The present invention also provides a method of typing a human subject as suffering from, or being at risk of suffering from, an inherited progressive brain disease selected from Parkinson Disease (PD), Parkinson Disease Dementia (PDD) and Dementia with Lewy bodies (DLB), the method comprising:

a) providing a sample comprising cells, or parts thereof, of said subject; b) determining in said cells or cell parts an amount and/or a level of activity of low density lipoprotein receptor-related protein 10 (LRP10) or the presence of one or more mutations in the LRP10 gene that affect stability or subcellular localization of the LRP10 protein;

c) correlating said determined amount and/or level of activity, or the presence of said one or more mutations, to a healthy control; and

d) typing the human subject as suffering from, or being at risk of suffering from, said inherited progressive brain disease if the determined amount and/or level of activity of LRP10 is reduced, when compared to the healthy control, or if said one or more mutations in the LRP10 gene is present.

A level of activity of low density lipoprotein receptor-related protein 10 (LRP10) may be determined by sequence analysis of a LRP 10-encoding gene, preferably including sequence analysis of one or more of human genomic coordinates 23346401, 23346689, 23345586, 23346192, 23345076, 23344860, 23346022, 23346023, 23346024 and 23344789 (Genome Reference Consortium Human Build 37 (GRCh37). This is possible, because the present inventors have found in patients suffering from an inherited progressive brain disease the existence of mutations in the LRP 10-encoding gene that result in coding variants or splice variants. In total, the inventors identified nine variants (A to I, as identified in Figure 1) of the protein associated with a reduced amount and/or a level of activity of the LRP10 protein. These mutations included missense mutations, wherein a nucleotide change results in a change of an amino acid residue in the protein product; frameshift mutations caused by insertion and/or deletion of one or more nucleotides such that the reading frame is altered; and alterations that affect splicing of the immature messenger RNA (mRNA) such that an exon is skipped and not included in the mature mRNA, or an aberrant exon is spliced into the mature mRNA. Thus, a reduced level of activity of low density lipoprotein receptor-related protein 10 (LRP10) is indicated when said sequence analysis indicates the presence of one or more mutations in the LRP10 gene. In particular, said mutations encode pathogenic variants of the LRP10 protein.

Pathogenic variants of the LRP10 protein include LRP10 protein with reduced stability or altered subcellular localization. As an alternative, or in addition, a level of activity of LRP10 may be determined by determining an amount and/or a level of protein activity of LRP10 in said cells or cell parts. Said amount and/or level of protein activity of LRP10 is preferably determined in cellular vesicles, preferably in exosomes. Said cellular vesicles, preferably exosomes, are preferably derived from brain cells, preferably from astrocytes and/or glial cells, blood cells or fibroblasts. Said cellular vesicles, preferably exosomes are preferably isolated from a bodily fluid, preferably selected from blood, saliva, tears, urine and cerebrospinal fluid.

The invention further provides a method of screening for a level of activity of LRP10, said method comprising a) providing a cell that expresses LRP10 and, optionally, alpha-synuclein, and b) determining a level of activity of LRP10, optionally by determining the amount of intra/extra cellular alpha-synuclein, more preferably a ratio of intra/extra cellular levels of alpha-synuclein.

The invention further provides a method of screening for a candidate compound for treating progressive brain disease, said method comprising a) providing a cell that expresses LRP10, b) adding one or more compounds to said cell, c) determining a level of LRP10 activity in said cell, preferably by determining the amount of extracellular alpha-synuclein, and d) designating said one or more compounds as candidate compound(s) for treating progressive brain disease, referred to herein as stimulator of LRP10, in case said LRP10 activity is increased. In a preferred method of the invention, a compound is identified that modulates the ratio of intra/extra cellular levels of alpha-synuclein.

In a further preferred method of the invention, a cell is provided that expresses a mutated LRP10 protein, preferably comprising one or more mutations at human genomic coordinates 23346401, 23346689, 23345586, 23346192,

23345076, 23344860, 23346022, 23346023, 23346024 and 23344789 (Genome

Reference Consortium Human Build 37 (GRCh37).

The invention further provides a method of treating a human subject suffering from, or being at risk of suffering from, a progressive brain disease, comprising providing a stimulator of LRP10, preferably a stimulator of LRP10- mediated modulation of intra/extra cellular levels of alpha-synuclein levels, and administering said stimulator of LRP10 to said human subject. Said stimulator of LRP10 preferably is an expression construct that expresses low density lipoprotein receptor-related protein 10 (LRP10), or a protein that is at least 70% identical to LRP10. Said stimulator of LRP10 preferably is provided to the brain.

The invention further provides a composition comprising a stimulator of LRP10 and a pharmaceutically acceptable excipient. FIGURE LEGENDS

Figure 1. LRP10 gene structure and protein structure. 1A: LRP10 gene structure; capital letters indicate the position of the nine identified variants A to I. IB: LRP10 protein structure; capital letters indicate the position of the seven coding variants identified (splicing variants C and F not shown); one variant (I) leads to frameshift and premature protein truncation, and nonsense-mediated mRNA decay. CUB = complement Clr/Cls, Uegf, Bmpl; LDLA = Low Density Lipoprotein Receptor Class A; TM = transmembrane domain; R-rich = Arginine- rich domain; P-rich = Proline -rich domain. 1C: Nucleotide sequence of a part of the LRP10-encoding gene. Indicated in upper case letters are exonic sequences, in lower case letters are intronic sequences. Italics denote untranslated regions. Underlined are the start codon (ATG) and stop codon (TGA). Underlined and in bold are pathogenic variants c.632dupT p.Ala212Serfs*17 (insertion of a T after coding DNA position 632), c.703C>T p. Arg235Cys (alteration of C into T nucleotide), c.919T>Ap.Tyr307Asn (alteration of T into A nucleotide), (two splicing variants at the same intronic nucleotide): c.l424+5delG (deletion of G nucleotide) and C.1424+5G>A (alteration of G into A nucleotide), c.l549_1551delAAT p.Asn517del (in frame deletion of nucleotides AAT), C.1598G>T p.Arg533Leu (alteration of G into T nucleotide), c.1807G>A p.Gly603Arg (alteration of G into A nucleotide), and c.2095C>T p.Pro699Ser (alteration of C into T nucleotide).

Figure 2 Pedigree of family 1 with PD: Pedigree and segregation of the LRP10 p.Gly603Arg variant. Black symbols denote affected individuals; grey symbols indicate individuals reported with PD by history, but not personally examined within this study; circles indicate women; squares indicate men; diamonds indicate gender- disguised individuals. The mean age at symptoms onset was 59.8 years (SD 8.7, range 46-73). Clinical details for each patient are reported in Table 3. M, heterozygous LRP10 p.Gly603Arg variant carrier; WT, homozygous wild type subject. The numbers below individual codes indicate ages at onset of symptoms (for patients), and age at last examination (for living unaffected carriers). The arrow indicates the proband.

Figure 3 Agarose gel analysis of the LRP10 transcripts. In comparison to normal controls, in patients carrying the c.l424+5delG or the C.1424+5G>A heterozygous mutation, a 1014-bp band becomes markedly more abundant, whereas the larger 1921-bp band, including part of the LRP10 transcript, is much decreased.

Figure 4 Effect of missense variants on LRP10 protein stability. HEK-293 cells expressing V5-tagged wildtype or LRP10 variants were treated with cycloheximide (CHX) for indicated times, followed by Western blot (WB) analysis. LRP10 protein levels were detected with anti-V5-tag antibody; Vinculin levels were used as loading controls. 4A: Representative WB images comparing wildtype, p.Arg533Leu, p.Tyr307Asn, and p.Gly603Arg expression levels. 4B: Representative WB images comparing wildtype, p.Asn517del, p.Arg235Cys, and p.Pro699Ser expression levels.

Figure 5 Effect of missense variants on LRP10 cell surface localization. The corrected total cell fluorescence (CTCF) of non-permeabilized cells labelled with intensities above background was quantified. Number of cells counted (n) for each condition is indicated. Median and 95% confidence interval are shown. Cells were counted from 3 independent experiments. Kruskal -Wallis and Dunn's multiple comparisons test was performed. Values passing the threshold for statistical significance of p<005 compared to wildtype are shown.

Figure 6 LRP10 markedly increases extracellular alpha -synuclein levels. WB analysis of extracellular and intracellular levels. BSA and GAPDH are shown as loading controls. HEK293T cells transfected with alpha-synuclein are indicated by arrows. GFP and LRP10-V5 are indicated.

Figure 7 Small molecules modulate LRP10 mediated increased extracellular alpha-synuclein levels. 7 A: WB analysis of extracellular (medium) and intracellular (cells) alpha-synuclein levels after treatment with small molecules, i.e., 1,2-Bis(2- aminophenoxy)e thane -N, N, N', N'- tetr a acetic acid tetrakis(acetoxymethyl ester) (BAPTA AM) and Ionomycin (calcium salt, CAS No. 56092-82-1), or control treatment (DMSO). HEK293T cells transfected with alpha-synuclein or LRP10-V5 have been indicated. Vinculin is shown as loading controls. 7B: Quantification of the effect of the small molecule treatment (BAPTA AM and Ionomycin vs. Control) on the ratio of extracellular/intracellular alpha-synuclein.

DETAILED DESCRIPTION OF THE INVENTION

(A) Definitions

The term“progressive brain disease”, as is used herein, refers to disease that results in progressive nervous system dysfunction. Such diseases are often associated with atrophy of the affected structures of the nervous system. They include diseases such as Alzheimer's Disease (AD) and other dementias,

Parkinson's Disease (PD), Parkinson Disease Dementia (PDD) and Dementia with Lewy bodies (DLB). The term progressive brain disease preferably refers to PD, PDD and DLB.

The term“low density lipoprotein receptor-related protein 10 (LRP10)”, as is used herein, refers to a single pass, type I transmembrane protein with Uniprot accession number Q7Z4F1. The LRP10 protein is a canonical protein of 713 amino acid residues, of which the N-terminal 16 residues provide a signal peptide (see Figure 1).

The term“LRPlO-encoding gene”, as is used herein, refers to a gene on chromosome 14 at position 14qll.2. The gene is transcribed between positions 23,340,822 and 23,350,789 (GRCh37.pl3 of the nucleotide sequence of human chromosome 14 with NCBI Reference Sequence: NC_000014.8. The gene has ENSEMBL accession number ENSG00000197324. The gene is also known as DKFZP564C 1940, LRP9 in mouse, MGC8675, MST087 and MSTP087.

As used herein, the term“alpha-synuclein encoding gene" refers to a gene on chromosome 4 at position 4q22.1. The gene is transcribed between positions 90,645,250 and 90,759,447 (minus DNA strand) (GRCh37/hg19). The gene, SNCA, has ENSEMBL accession number ENSG00000145335. The gene is also known as SNCA, NACP, PARK1, PARK4, and PD1. The term gene, as used herein, includes upstream and downstream

chromosomal sequences that are relevant for expression of the protein in relevant cells. Such regulatory sequences may be located up to 100kb upstream and/or downstream of the transcribed region.

The terms“alpha-synuclein” and“alpha-synuclein protein”, as are used herein, refer to a protein that is encoded by the alpha-synuclein gene. The protein has UniProt accession number P37840. The terms alpha-synuclein” and“alpha- synuclein protein include reference to monomers, oligomers, and aggregates of alpha-synuclein.

The term“typing”, as is used herein, refers to diagnosing a human subject as suffering from a progressive brain disease, or assessing a risk for a subject of suffering from a progressive brain disease. Said typing is intended to provide diagnostic and/or prognostic information to aid in clinical evaluation of the subject and the disease. For example, said typing may assist a classification of a subject suffering from a progressive brain disease or being at risk of suffering therefrom, and/or may assist in designing an optimal therapeutic regimen for the subject.

The term“level of activity of LRP10”, as is used herein, refers to an activity of LRP10 that can be measured and, preferably, quantified such as the

internalization of lipophilic, molecules including the uptake of lipoprotein apolipoprotein E (ApoE) in liver cells. The level of activity of LRP10 specifically refers to the amount of intra/extra cellular alpha- synuclein, preferably the ratio of intra/extra cellular levels of alpha-synuclein, that can be determined in cells that express both LRP10 and alpha-synuclein.

The term“bodily fluid”, as used herein, refers to blood (or a fraction of blood such as plasma or serum), lymph, mucus, tears, saliva, sputum, urine, semen, stool, CSF (cerebrospinal fluid), breast milk and ascites fluid. Preferably, the bodily fluid is selected from cerebrospinal fluid, tears, saliva, blood and urine.

The term "nucleic acid", as used herein, includes reference to a

deoxyribonucleotide or ribonucleotide polymer in either single -or double -stranded form, and unless otherwise indicated, encompasses known analogues that can hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e. g., peptide nucleic acids). The term“ribonucleic acid or RNA”, as is used herein, refers to protein encoding ribonucleic acid or a non-protein encoding ribonucleic acid such as rRNA and miRNA.

The term“mutation”, as is used herein, refers to an alteration that alters a native sequence by displacement, addition, deletion, insertion, cross-linking, or other substitution of one or more nucleotides of the native sequence, including naturally occurring splice variants. In particular, the term mutation refers to an alteration that has an effect on the coding sequence of a gene, such as a missense mutation, a nucleotide change that results in a change of an amino acid residue, a frameshift mutation caused by insertion and/or deletion of one or more nucleotides such that the reading frame is altered, and an alteration that affects splicing of the immature messenger RNA (mRNA) such that an exon is skipped and not included in the mature mRNA, or an aberrant exon is spliced into the mature mRNA.

The term“replacement” or“replaced”, as used herein, refers to the

substitution of one or more amino acid residues, thereby altering the amino acid sequence, but not the number of amino acid residues. A replacement is the consequence of the deletion of an amino acid residue followed by the insertion of a different amino acid residue at the same position.

The term“deletion” or“deleted”, as used herein, refers to deleting one or more amino acid residues of a protein, thereby reducing the number of amino acid residues of said protein.

The term "amplified", as used herein, refers to the construction of multiple copies of a nucleic acid sequence and/or multiple copies complementary to the nucleic acid sequence using at least one of the nucleic acid sequences as a template. Amplification systems include the polymerase chain reaction (PCR) system, ligase chain reaction (LCR) system, nucleic acid sequence based amplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicase systems, transcription-based amplification system (TAS), and strand displacement amplification (SDA). See, e. g., Diagnostic Molecular Microbiology. Principles and Applications, D. H. Persing et al., Ed., American Society for Microbiology, Washington, D. C. (1993). The product of amplification is termed an amplicon.

The term "hybrid" refers to a double-stranded nucleic acid molecule, or duplex, formed by hydrogen bonding between complementary nucleotides. The terms "hybridize" or "anneal" refer to the process by which single strands of nucleic acid sequences form double -helical segments through hydrogen bonding between complementary nucleotides.

The term "oligonucleotide", as used herein, refers to a short sequence of nucleotide monomers (usually 6 to 100 nucleotides) joined by phosphorous linkages (e.g., phosphodiester, alkyl and aryl-phosphate, phosphorothioate), or non- phosphorous linkages (e.g., peptide, sulfamate and others). An oligonucleotide may contain modified nucleotides having modified bases (e.g., 5-methyl cytosine) and modified sugar groups (e.g., 2'-0-methyl ribosyl, 2'-0-methoxyethyl ribosyl, 2'- fluoro ribosyl, 2'-amino ribosyl, and the like). Oligonucleotides may be naturally- occurring or synthetic molecules of double- and single-stranded DNA and double - and single-stranded RNA with circular, branched or linear shapes and optionally including domains capable of forming stable secondary structures (e.g., stem-and- loop and loop-stem-loop structures).

The term "primer" as used herein refers to an oligonucleotide which is capable of annealing to the amplification target, also termed template, allowing a DNA polymerase to attach thereby serving as a point of initiation of DNA synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a template nucleic acid strand is induced, i.e., in the presence of nucleotides and an agent for polymerization such as DNA polymerase and at a suitable temperature and pH. A primer preferably is single stranded for maximum efficiency in amplification. Preferably, the primer is an oligodeoxy ribonucleotide. The primer preferably is sufficiently long to prime the synthesis of extension products. The exact length of a primer will depend on many factors, including temperature and source of primer. A "pair of bi-directional primers" as used herein refers to one forward and one reverse primer as commonly used in the art of DNA amplification such as in PCR amplification.

The term "probe" refers to a single-stranded oligonucleotide sequence that will recognize and form a hydrogen-bonded duplex with a complementary sequence in a target nucleic acid sequence analyte or its cDNA derivative, such as an amplicon.

The terms "stringency" and "stringent hybridization conditions" refer to hybridization conditions that affect the stability of hybrids, e.g., temperature, salt concentration, pH, formamide concentration and the like. These conditions are empirically optimised to maximize specific binding and minimize non-specific binding of primer or probe to its target nucleic acid sequence. The terms as used include reference to conditions under which a probe or primer will hybridise to its target sequence, to a detectably greater degree than other sequences (e.g. at least 2-fold over background). Stringent conditions are sequence dependent and will be different in different circumstances. Longer sequences hybridise specifically at higher temperatures. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridises to a perfectly matched probe or primer. Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M Na+ ion, typically about 0.01 to 1.0 M Na+ ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes or primers (e.g. 10 to 50 nucleotides) and at least about 60°C for long probes or primers (e.g. greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringent conditions or "conditions of reduced stringency" include hybridization with a buffer solution of 30% formamide, 1 M NaCl, 1% SDS at 37°C and a wash in 2x SSC at 40°C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in O.lx SSC at 60°C. Hybridization procedures are well known in the art and are described in e.g. Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons Inc., 1994.

The term“% identical” or“% identity”, as is used herein, refers to a nucleic acid or amino acid sequence identity between two nucleic acid or amino acid molecules, expressed as a percentage of the total length of the two molecules.

Sequence identity is determined by comparing the identity of individual nucleic acid residues or amino acid residues of one molecule to the corresponding residues in another molecule.

The term“stimulator of LRP10 activity”, as is used herein, refers to a molecule that activates a level of LRP10 activity in a cell, such as the

internalization of lipophilic molecules such as lipoprotein apolipoprotein E (ApoE). A stimulator of LRP10 activity preferably modulates the intra/extra cellular levels of alpha-synuclein, preferably the ratio of intra/extra cellular levels of alpha- synuclein. A stimulator of LRP10 activity may enhance a level of extracellular alpha-synuclein in one cell type, such as in HEK293 cells, while lowering a level of extracellular alpha-synuclein, for example by promoting the uptake of alpha- synuclein, in another cell type such as in astrocytes and/or glial cells.

As used herein, the term "treatment" or "treating" refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and may be performed during the course of clinical pathology. Desirable effects of the treatment include preventing occurrence or recurrence of the illness, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the illness, and/or amelioration or palliation of the state of the illness. In the present invention, "treatment" or "treating" is understood to mean amelioration or palliation of a human suffering from a progressive brain disease by administering a pharmaceutical composition comprising a stimulator of LRP10.

(B) Methods of typing

The present invention provides a method of typing a human subject by providing a relevant sample comprising cells, or parts thereof, of said subject, determining in said sample an amount and/or a level of activity of low density lipoprotein receptor-related protein 10 (LRP10), correlating said determined amount and/or level of activity to a control, and typing the human subject as suffering from, or being at risk of suffering from, a progressive brain disease if the determined amount and/or level of activity of LRP10 is reduced, when compared to the control.

Typing of a sample comprises determination of an amount and/or a level of activity of low density lipoprotein receptor-related protein 10 (LRP10) in cells or parts thereof from a subject. Said cell preferably comprises one or more blood cells and/or neuronal cells or parts thereof.

In an embodiment, an amount and/or a level of activity of LRP10 is determined by determining a gene copy number of LRP10, by epigenetic analysis of LRP10, preferably including a genomic region surrounding LRP10, such as determining methylation patterns, histone acetylation, etc., as is known to a person skilled in the art.

In an embodiment, an amount and/or a level of activity of LRP10 is determined by determining a sequence of a gene encoding LRP10 or parts thereof, and/or of the LRP10 protein or parts thereof. As is indicated herein above, the gene is transcribed between positions 23,340,822 and 23,350,789 (GRCh37.p13) of NCBI Reference Sequence: NC_000014.8, but regulatory sequences may be located up to 100kb upstream and/or downstream of the transcribed region.

A nucleotide sequence may be determined of a complete gene encoding LRP10, including upstream and/or downstream regulatory sequence, or of parts thereof such as, for example, exonic parts of the gene, a messenger RNA that is transcribed from the gene, and/or hotspots in which alterations are expected to occur. A nucleotide sequence of said gene or parts thereof may be determined after isolating of a nucleic acid molecule comprising the gene or parts thereof from a cell of the subject, preferably a blood cell and/or neuronal cell. As an alternative, the gene or parts thereof may be amplified from a cell of the subject, preferably a blood cell and/or neuronal cell, followed by determining the nucleic acid sequence of the one or more amplicons.

Said amplicons preferably include genomic coordinates 23346401, 23346689, 23345586, 23346192, 23345076, 23344860, 23346022, 23346023, 23346024 and 23344789 of NC_000014.8.

A nucleotide sequence of a gene encoding LRP10, or parts thereof may be determined by any method known in the art, including chemical sequencing methods and chain termination methods involving primer-initiated synthesis by addition of deoxynucleoside triphosphates, a DNA polymerase and chain

terminating dideoxy NTPs. Preferred sequence methods include high throughput sequencing methods such as single-molecule real-time sequencing (Pacific

Biosciences), pyrosequencing (454 Life Sciences), sequencing by synthesis

(Illumina) and nanopore sequencing, including solid state nanopore sequencing and biological nanopore sequencing (e.g., Roche and Oxford Nanopore Technologies).

An amino acid sequence of LRP10 protein or parts thereof may be determined by hydrolysis of the protein or a part thereof, followed by separation and identification of the resulting fragments. Said hydrolysis may be performed by incubating a sample of the protein in a strong acid, for example in 6 M hydrochloric acid at an elevated temperature, for example at 100-110 °C, for a prolonged period of time, for example for 24 hours or longer. Reagents such as thiol or phenol may be added to protect tryptophan and tyrosine from attack by chlorine. As an

alternative, or in addition, hydrolysis may be performed by endopeptidases such as trypsin or pepsin or by chemical reagents such as cyanogen bromide. Different enzymes give different cleavage patterns, and the overlap between fragments can be used to construct an overall sequence.

An amino acid sequence of LRP10 protein and/or parts thereof may be determined by Edman degradation employing, for example a Beckman-Coulter Porton LF3000G protein sequencing machine, and/or by mass spectrometry using, for example, high performance liquid chromatography (HPLC) or ultra-high performance liquid chromatography (UHPLC), coupled to tandem mass

spectrometry (LC-MS/MS) in a positive electrospray ionization mode. LC-MS/MS analysis may be performed, for example, by employing a high end UHPLC chromatographic system coupled to a triple - quadrupole mass-spectrometer.

As an alternative, or in addition, sequencing a protein or part thereof may be performed from its C-terminus using enzymes called carboxypeptidases, which remove individual C-terminal amino acids. For example, carboxypeptidase B can release the amino acids arginine and lysine from the C-terminus of a protein, while carboxypeptidase A can cleave off all other amino acid residues but not arginine, lysine, or proline.

In an embodiment, an amount and/or a level of activity of LRP10 is determined by determining the amount of LRP10 mRNA. For this, said mRNA preferably is converted into complement DNA (cDNA) using a RNA-dependent

DNA polymerase or reverse transcriptase such as a HIV-1 reverse transcriptase, a M-MLV reverse transcriptase and/or an AMV reverse transcriptase, or a derivative of one of these reverse transcriptases such as a recombinant M-MuLV reverse transcriptase with reduced RNase H activity and increased thermostability (ProtoScript® II Reverse Transcriptase, New England Biolabs).

The resultant cDNA is subsequently amplified employing a amplification technique such as Polymerase Chain Reaction (PCR), rolling circle amplification, nucleic acid sequence-based amplification, transcription mediated amplification, and linear RNA amplification. A preferred amplification method is PCR, especially real-time PCR.

PCR is a technology that relies on thermal cycling, consisting of cycles of repeated heating and cooling of a reaction for DNA melting and enzymatic replication of the DNA. Primers containing sequences that specifically hybridize to the target region, and a DNA polymerase are key components to enable selective and repeated amplification. As PCR progresses, the amplified DNA product that is generated is itself used as a template for replication, resulting in a chain reaction in which the DNA template is exponentially amplified,

A preferred DNA polymerase is a thermostable polymerase, preferably a thermostable recombinant polymerase. Preferred commercially available DNA polymerases include AptaTaq Fast DNA Polymerase and LightCycler® FastStart Enzyme (Roche Diagnostics, Almere, The Netherlands).

Real-time PCR, also called quantitative PCR (qPCR), is a technique which is used to amplify and simultaneously quantify a template DNA molecule. The detection of the amplification products can in principle be accomplished by any suitable method known in the art. The amplified products may be directly stained or labelled with radioactive labels, antibodies, luminescent dyes, fluorescent dyes, or enzyme reagents. Direct DNA stains include for example intercalating dyes such as acridine orange, ethidium bromide, ethidium mono azide or Hoechst dyes.

Alternatively, the amplified product may be detected by incorporation of labelled dNTP bases into the synthesized DNA fragments. Detection labels which may be associated with nucleotide bases include, for example, fluorescein, cyanine dye and BrdUrd.

When using for example Scorpion primers or a probe-based detection system, a primer or the probe is preferably labelled with a detectable label, preferably a fluorescent label. Preferred labels for use in this invention comprise fluorescent labels, preferably selected from Atto425 (ATTO-TEC GmbH, Siegen, Germany), Atto 647N (ATTO-TEC GmbH, Siegen, Germany), YakimaYellow (Epoch

Biosciences Inc, Bothell, WA, USA), CalGlO (BioSearch Technologies, Petaluma,

CA, USA), Cal635 (BioSearch Technologies, Petaluma, CA, USA), FAM (Thermo Fisher Scientific Inc., Waltham, MA USA), TET (Thermo Fisher Scientific Inc., Waltham, MA USA), HEX ((Thermo Fisher Scientific Inc., Waltham, MA USA), cyanine dyes such as Cy5, Cy5.5, Cy3, Cy3.5, Cy7 (Thermo Fisher Scientific Inc., Waltham, MA USA), Alexa dyes (Thermo Fisher Scientific Inc., Waltham, MA USA), Tamra (Thermo Fisher Scientific Inc., Waltham, MA USA), ROX (Thermo Fisher Scientific Inc., Waltham, MA USA), JOE (Thermo Fisher Scientific Inc., Waltham, MA USA), fluorescein isothiocyanate (FITC, Thermo Fisher Scientific Inc., Waltham, MA USA), and tetramethylrhodamine (TRITC, Thermo Fisher Scientific Inc., Waltham, MA USA). A probe is preferably labeled at the 5’ end with a detectable label, preferably a fluorescent label.

A primer such as a Scorpion primer, or a probe preferably has a fluorescent label at one end and a quencher of fluorescence at the opposite end of the probe. The close proximity of the reporter to the quencher prevents detection of its fluorescence; breakdown of the probe by the 5' to 3' exonuclease activity of polymerase breaks the reporter-quencher proximity and thus allows unquenched emission of fluorescence, which can be detected after excitation with a laser. An increase in the product targeted by the reporter probe at each PCR cycle therefore causes a proportional increase in fluorescence due to the breakdown of the probe and release of the reporter. Quenchers, for example tetramethylrhodamine

TAMRA, dihydrocyclopyrroloindole tripeptide minor groove binder, are known in the art.

Preferred quenchers are Black Hole Quencher®-1 (BHQ1) and BHQ2, which are available from Biosearch Technologies, Petaluma, CA, USA). The BHQ1 dark quencher has strong absorption from 480 nm to 580 nm, which provides excellent quenching of fluorophores that fluoresce in this range, such as FAM, TET, CAL Fluor® Gold 540, JOE, HEX, CAL Fluor Orange 560, and Quasar® 570 dyes. The BHQ2 dark quencher has strong absorption from 599 nm to 670 nm, which provides excellent quenching of fluorophores that fluoresce in this range, such as Quasar® 570, TAMRA, CAL Fluor® Red 590, CAL Fluor Red 610, ROX, CAL Fluor Red 635, Pulsar® 650, Quasar 670 and Quasar 705 dyes. BHQ1 and BHQ2 may quench fluorescence by both FRET and static quenching mechanisms.

As an alternative, or in addition, an amount of LRP10 mRNA is determined by high-throughput next- generation sequencing (NGS) technologies. These technologies include cluster amplification of DNA strands prior to fluorescent or potentiometric sequencing, as are available from ThermoFisher Scientific (Ion Torrent™ Personal Genome Machine™ System) and from Illumina (e.g., NextSeq® and HiSeq®). These technologies also include single molecule sequencing technologies with specially designed fluorescent detection systems (Zero-mode Waveguides) and nanopore sequencing using exonuclease or DNA polymerase activities (Roche and Oxford Nanopore Technologies). An amount of LRP10 mRNA is determined by providing a ratio of the number of LRP10 mRNA or cDNA molecules to the total number of mRNA or cDNA molecules that were sequenced.

In one aspect, an amount and/or a level of activity of LRP10 is determined by determining the amount and/or activity of LRP10 protein. For this, a sample comprising proteins from relevant cells of the subject is provided. For example, a sample comprising protein may be obtained from a tissue sample or a biopsy sample comprising cells from the subject, for example, blood cells, including circulating stem cells, keratinocytes and/or fibroblasts. The surgical step of removing a relevant tissue sample from a subject is not part of a method according to the invention. If needed, the cells, e.g. the blood cells or fibroblasts, are differentiated into neuronal cells or neuronal-like cells, prior to determining an amount and/or a level of activity of LRP10. Said sample can be obtained in numerous ways, as is known to a skilled person. For example, the sample can be freshly prepared from cells or a tissue sample at the moment of harvesting, or it can be prepared from a sample that was stored at -70°C until processed for sample preparation. Alternatively, a tissue sample may be stored under conditions that preserve the quality of the protein such as fixation using e.g. formaline.

As an alternative, or in addition, an amount and/or a level of activity of LRP10 is determined after expression of a LRP10 mRNA from a subject, or cDNA generated from said LRP10 mRNA in a tester cell. Methods for isolating mRNA, converting it into cDNA and expressing LRP10 protein in a cell, preferably a eukaryotic cell, are known in the art.

Due to the fact that LRP10 is present in cellular vesicles, including exosomes, an amount and/or a level of activity of LRP10 is preferably determined in exosomes. Said exosomes preferably are derived from a relevant cell such as a brain cell, especially astrocyte and/or glial cell, a blood cell or a fibroblast. Said exosomes preferably are isolated from a bodily fluid, more preferably from cerebrospinal fluid, blood and/or urine. Methods for isolation of exosomes are known to a person skilled in the art, and include differential centrifugation, density gradient centrifugation, size exclusion chromatography, filtration, polymer-based precipitation, sieving and immunological separation techniques such as magnetic beads. A method may be applied that allows isolation of all exosomes or of selective subtypes of exosomes. Kits for isolation of exosomes and other extracellular vesicles from a bodily fluid are commercially available, including“exoEasy Maxi Kit” (Qiagen) and Exo-spin Purification (Cell Guidance Systems Ltd, Cambridge, UK).

An amount of LRP10 in a relevant cell or part thereof, preferably in a brain cell, a blood cell or a fibroblast, or exosome derived from said cell, may be determined by, for example, two dimensional gel electrophoresis, multidimensional protein identification technology, ELISA, liquid chromatography-mass

spectrometry (LC-MS), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). A further preferred method includes the use of differentiating antibodies that interact with either a non-mutated normal form, or with a mutated variant form of LRP10.

A level of activity of LRP10 in a relevant cell or part thereof, may be determined by adding lipoprotein APOE to the cell or part thereof expressing LRP10 and determining the amount of internalized lipoprotein APOE. As an alternative, or in addition, a level of activity of LRP10 may be determined by adding alpha-synuclein to the cell or part thereof and determining the amount of extracellular alpha-synuclein. Said APOE and/or alpha-synuclein preferably carries a label, such as a fluorescent label, a (radio)isotope label, and/or a paramagnetic label. A (radio)isotope label is preferably a radioactive label.

A level of expression and/or a level of activity of LRP10 preferably is normalized, meaning that variation due to, for example, differences in sample size, the use of different analytical platforms, and/or different laboratories is excluded as much as possible. As is known to a person skilled in the art, expression levels of a protein termed“A” in different samples are often compared by determining a relative level of expression of protein A to a reference protein B in all samples.

Provided that reference protein B is expressed at the same level in all sample, the relative expression of protein A/protein B provides a normalized estimate of the level of expression of A in the samples. Similarly, the level of activity of LRP10 in different samples may be compared by normalizing the level of expression of LRP10 in the samples.

A level of expression and/or a level of activity of LRP10 as determined in relative cells or parts thereof of a subject, is correlated to a determined amount and/or level of activity in a control. Said control preferably is or comprises a level of expression and/or a level of activity of LRP10 in an individual that is not suffering from a progressive brain disease, more preferably is or comprises an average level of expression and/or an average level of activity of LRP10 in at least 5 individuals that are not suffering from a progressive brain disease. The average level of expression and/or an average level of activity of LRP10 preferably is determined in, for example, 5-100 individuals, such as between 10 and 20 individuals, including about 15 individuals. As an alternative, or in addition, said control preferably is or comprises a level of expression and/or a level of activity of LRP10 in an individual that is suffering from a progressive brain disease such as Parkinson’s disease, more preferably is or comprises an average level of expression and/or an average level of activity of LRP10 in at least 10 individuals that are suffering from a progressive brain disease.

Typing of a sample comprising neuronal cells or parts thereof can be performed in various ways. In one method, a coefficient is determined that is a measure of a similarity or dissimilarity of a sample with a previously established level of expression and/or a level of activity of LRP10 that is specific of a certain cell type, tissue, disease state or any other interesting biological or clinically- relevant sample or group of samples. A specific level of expression and/or a level of activity of LRP10 in a specific cell type, tissue, disease state or any other interesting biological or clinically-relevant sample or group of samples may be termed a“template”. Typing of a sample can be based on its (dis)similarity to a template or based on multiple templates such as a control template from

individuals that are not suffering from a progressive brain disease, and a control template from individuals that are suffering from a progressive brain disease. In addition, control templates may be generated from individuals that are suffering, for example, from Alzheimer disease, Parkinson Disease Dementia (PDD) and Dementia with Lewy bodies (DLB). A number of different coefficients can be used for determining a correlation between the level of expression and/or a level of activity of LRP10 in a sample from a subject and a reference sample. Preferred methods are parametric methods which assume a normal distribution of the data. Preferred methods comprise cosine- angle, un-centered correlation and, cosine correlation (Fan et al., Conf Proc IEEE Eng Med Biol Soc. 5:4810-3 (2005)) and Pearson product-moment correlation coefficient, which is obtained by dividing the covariance of two variables by the product of their standard deviations.

Preferably, said correlation with a template is used to produce an overall similarity score. A similarity score is a measure of the average correlation of a level of expression and/or a level of activity of LRP10 in a sample from a subject and a template. Said similarity score can, for example, be a numerical value between +1, indicative of a high correlation between the level of expression and/or a level of activity of LRP10 in a sample of said subject and said template, and -1, which is indicative of an inverse correlation. Preferably, an arbitrary threshold is used to type a sample A similarity score is preferably displayed or outputted to a user interface device, a computer readable storage medium, or a local or remote computer system.

A method according to the invention may further comprise assessment of clinical information, such as tremor, bradykinesia, muscle stiffness, posture and balance, unconscious movements, changes in speech and writing, and family history.

(C) Methods of screening

The invention further provides a method of screening for a level of activity of low density lipoprotein receptor-related protein 10 (LRP10), comprising providing a cell that expresses LRP10 and alpha-synuclein, and determining the amount of intra/extra cellular alpha-synuclein, more preferably a ratio of intra/extra cellular alpha-synuclein.

A cell, preferably a eukaryotic cell, may endogenously express LRP10 and/or alpha-synuclein. Eukaryotic cells that express endogenous levels of LRP10 and alpha-synuclein include HeLa human cervical cancer cells (ATCC® CCL-2™), HuTu-80 human duodenum carcinoma cells (ATCC® HTB-40™) and SH-SY5Y (human bone marrow neuroblast cells (ATCC ® CRL-2266™ ). LRP10 knockout cell lines may be generated using CRISPR-Cas9 methods.

Said cell may be any cell of the body, preferably a cell that expresses an abundant level of LRP10, including a blood cell, fibroblast, or brain cell, preferably an astrocyte and/or glial cell. Said cell includes a cell from a human induced pluripotent stem cell (iPSC) cell line (Vanhauwaert et al, 2017. EMBO J 36: 1392- 411) and a neural progenitor cell derived from iPSC colonies using known protocols (Reinhardt et al., 2013. PLoS One 8: e59252). Said iPSC cell lines preferably are derived from PD, PDD and DLB patients, comprising mutations in the LRP10 gene. Said mutations preferably include one or more mutations at genomic coordinates 23346401, 23346689, 23346192, 23345076, 23345586, 23344860, 23346022, 23346023, 23346024 and 23344789. If required, alterations of the endogenous LRP10 gene and/or the alpha-synuclein gene may be generated using CRISPR-Cas9 methods.

The amount of intra/extra cellular alpha-synuclein may be determined by any method known in the art that is suited for quantification of protein levels, including two dimensional gel electrophoresis, multidimensional protein

identification technology, ELISA, liquid chromatography-mass spectrometry (LC- MS), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF).

LRP10 and/or alpha-synuclein may be provided exogenously to a cell, preferably a eukaryotic cell, for example by transfecting or infecting the eukaryotic cell with an expression vector that encodes LRP10 and/or alpha-synuclein. The expression vector may be a plasmid that encodes LRP10 and/or alpha-synuclein. Suitable plasmids are, for example, pCAGGS, and pcDNA. Said expression vector preferably is a recombinant virus or viral vector that encodes LRP10 and/or alpha- synuclein. A suitable virus or viral vector is, for example, a replication defective retroviral vector such as a lentiviral vector, for example a HIV-based vector or an EIAV-based vector, or a replication defective MMLV-based vector. A further suitable virus or viral vector is provided by a replication defective adenoviral vector and/or an adenovirus - associate d viral vector. A further preferred expression vector is a herpes simplex virus-based vector that is able to transduce neuronal cells. A cell that is exogenously provided with LRP10 and/or alpha-synuclein may be any eukaryotic cell or cell line, including but not limited to Baby Hamster Kidney cells, Human Embryonic Kidney cells such as HEK293 and freestyle HEK293FTM cells (ThermoFisher Scientific), VERO cells, MDCK cells, CHO cells, HeLa and PER.C6 cells (Fallaux, F. J. et al. 1998. Hum Gene Ther 9: 1909-1917). Preferred cells are Human Embryonic Kidney cells such as HEK293 and freestyle HEK293FTM cells.

An exogenously provided LRP10 and/or alpha- synuclein may comprise at least one tag at the N- terminus and/or C-terminus of the protein. Said at least one tag, preferably repeats of a tag such as a tandem repeat, preferably is selected from HIS, CBP, CYD, Strep II, V5, FLAG and heavy chain of protein C peptide tags. A preferred tag is the V5 tag, comprising 14 amino acids (GKPIPNPLLGLDST), but which may be used with a shorter 9-amino acid (IPNPLLGLD) sequence. Said at least one tag sequence may be separated from the protein by a recognition and cleavage sequence for an endoprotease. Said endoprotease preferably is a ubiquitous endoprotease such as a subtilisin family member. Said recognition sequence may comprise two adjacent basic amino acid residues such as, for example, the amino acid sequence KR and/or RK. As an alternative, said endoprotease is enterokinase, having the recognition/cleavage sequence

DDDDK | X, and/or a TEV protease with a preferred recognition/cleavage sequence E(N/X)LYFQ | (S,G,A), where X could be any amino acid residue. In addition any other endoprotease that is known in the art to be suitable for this purpose may be employed.

Further provided is a method of screening for a compound that modulates LRP10 activity comprises (a) contacting a compound with LRP10, (b) determining a binding affinity of the compound to LRP10, (c) contacting a population of mammalian cells, preferably human neuronal cells, expressing said LRP10 with the compound that exhibits a binding affinity of at most 10 micromolar, and (d) identifying a compound that activates an activity of LRP10 in the cells.

Methods for screening compounds by screening a library of compound are known in the art. For example, a wide variety of NMR-based methods are available to rapidly screen libraries of small compounds for binding to protein targets (Hajduk, P. J., et al. Quarterly Reviews of Biophysics, 1999. 32 (3): 211-40). The methods are amenable to automated, cost-effective high throughput screening of chemical libraries for lead compounds. Identified reagents find use in the pharmaceutical industries for human trials; for example, the reagents may be derivatized and rescreened in in vitro and in vivo assays to optimize activity and minimize toxicity for pharmaceutical development.

Said activation of LRP10 preferably is selective over activation of other low- density lipoprotein receptor-related proteins, whereby the term selective indicates that the IC50 for activation of LRP10 is more than 2x lower than the IC50 for activation of another low-density lipoprotein receptor-related protein, preferably said IC50 for activation of LRP10 is more than 4x lower than the IC50 for activation of another low-density lipoprotein receptor-related protein, more preferably more than lOx lower.

The invention further provides a method of screening for a compound that modulates LRP10 activity, preferably LRPlO-mediated modulation of intra/extra cellular alpha- synuclein, comprising providing a cell that expresses LRP10, adding one or more compounds to said cell, and determining LRP10 activity after addition of the one or more compounds.

As is indicated herein above, a level of activity of LRP10 may be determined by adding lipoprotein APOE to the cell or part thereof and determining the amount of internalized lipoprotein APOE. As an alternative, or in addition, a level of activity of LRP10 may be determined by adding alpha-synuclein to the cell or part thereof and determining the amount of intra/extra cellular alpha- synuclein, preferably a ratio of intra/extra cellular alpha-synuclein. Said APOE and/or alpha- synuclein preferably carries a label, such as a fluorescent label and/or a

(radio)isotope label. A (radio)isotope label is preferably a radioactive label.

Said one or more compound preferably are added to separate wells

comprising cells that express LRP10. Said wells preferably are wells of a multi-well plate such as a 96 well plate, a 192 well plate, a 384 well plate and/or a 1536 well plate. Activation of LRP10 in a cell by a compound will result in increased internalization of lipoprotein APOE or modulation of the ratio of intra/extra cellular alpha- synuclein, when compared to a cell in which LRP10 is not activated by a compound. A comparison of activities in different wells, to which different compounds and/or different concentrations of a compound are added, will result in the identification of one or more compounds that activate LRP10.

Said library of compounds may comprise a dedicated library of 100-200 compounds whose structures are based on common structural elements of LRP10- interacting molecules such as APOE and/or alpha-synuclein. Larger libraries of up to 2000 compounds may be designed to cover a wider range of chemical structures with relatively low formula weight (100-200Da) and composition of functional groups that may interact with LRP10. Still larger libraries of up to 2 million drug like chemicals may be generated and screened using high throughput screening assays.

Said one or more compounds preferably is present amongst 1600 FDA approved drugs, as provided by Pharmakon 1600, Microsource Discovery Systems Inc. USA). In addition, compounds may be screened that have been identified through a connectivity mapping approach focusing on compounds that show an opposite effect on 1000 landmark gene expression data in comparison to the effect of LRP10 knockdown on the same landmark genes in several cell lines

(https://clue.io/). The Connectivity Map, or CMap, is a resource that uses transcriptional expression data to probe relationships between diseases, cell physiology, and therapeutics. The changes in gene expression, or "signatures," that arise from a disease, genetic perturbation (knockdown or overexpression of a gene) or treatment with a small molecule are compared for similarity to all

perturbational signatures in the database. Thus, perturbations that elicit highly similar, or highly dissimilar, expression signatures in comparison to LRP10, are termed "connected"; their related transcriptional effects suggest they confer related physiological effects on the cell.

(D) Methods of treatment

The invention further provides a method of treating a human subject suffering from, or being at risk of suffering from, a progressive brain disease, comprising providing a stimulator of LRP10 activity, preferably a stimulator of LRPlO-mediated modulation of the ratio of extra/intra cellular alpha-synuclein levels, and administering said stimulator of LRP10 to said human subject. A preferred stimulator of LRP10 is an expression construct that expresses low density lipoprotein receptor-related protein 10 (LRP10) itself, or a protein that is at least 70%, more preferred at least 80%, at least 90%, at least 95%, at least 99% identical to LRP10 over the entire length of the gene or protein. Said expression construct preferably is a vector, preferably a viral vector, encoding

LRP10 or a protein that is at least 70%, more preferred at least 80%, at least 90%, at least 95%, at least 99% identical to LRP10. Said vector preferably additionally comprises means for high expression levels such as strong promoters, for example of viral origin (e.g., human cytomegalovirus) or promoters derived from genes that are highly expressed in a cell such as a human neuronal cell (Running Deer and Allison, 2004. Biotechnol Prog 20: 880-889; US patent No: 5888809).

Said vector preferably is a viral vector, preferably a viral vector that is able to transduce human cells.

In one embodiment, said viral vector is a retroviral-based vector such as a lentivirus -based vector such as a human immunodeficiency virus-based vector, or a gamma-retro virus-based vector such as a vector based on Moloney Murine

Leukemia Virus (MoMLV), Spleen-Focus Forming Virus (SFFV),

Myeloproliferative Sarcoma Virus (MPSV) or on Murine Stem Cell Virus (MSCV).

A preferred retroviral vector is the SFG gamma retroviral vector (Riviere et al., 1995. PNAS 92: 6733-6737), or a lentivirus -based vector such as a human immunodeficiency virus-based vector.

Retroviruses, including a gamma-retrovirus-based vector, can be packaged in a suitable complementing cell that provides Group Antigens polyprotein (Gag)- Polymerase (Pol) and/or Envelop (Env) proteins. Suitable packaging cells are human embryonic kidney derived 293T cells, Phoenix cells (Swift et al., 2001. Curr Protoc Immunol, Chapter 10: Unit 10 17C), PG13 cells (Loew et al., 2010. Gene Therapy 17: 272-280), and Flp293A cells (Schucht et al., 2006. Mol Ther 14: 285- 92).

Said viral vector preferably is a recombinant adeno-associated viral vector or a herpes simplex virus-based vector (Choudhury et al., 2017. Neuropharmacology 120: 63-80). Replication- defective HSV vectors have been employed in Phase I-II human trials and have been explored as delivery vehicles for disorders such as pain, neuropathy, and other neurodegenerative conditions. Viral expression in vivo preferably is directed at targeting neuronal cells in the brain, preferably neuronal cells in the olfactory bulb, dorsal motor nucleus of the vagus, and substantia nigra pars comp acta.

As an alternative, non-viral gene therapy may be used for in vivo expression of LRP10 or a protein that is at least 70%, more preferred at least 80%, at least 90%, at least 95%, at least 99%, identical to LRP10 in relevant cells such as neuronal cells. Non-viral delivery may be provided by, for example, nude DNA, liposomes, polymerizers and molecular conjugates, as is known to a skilled person. Minicircle DNA vectors free of plasmid bacterial DNA sequences may be generated and may express a nucleic acid encoding LRP10 or a protein that is at least 70% identical to LRP10 at high levels in vivo.

A stimulator of LRP10 such as BAPTA AM preferably is provided directly to the brain, preferably to the substantia nigra.

The invention further provides a stimulator of LRP10, preferably a stimulator of LRPlO-mediated modulation of the ratio of intra/extra cellular alpha-synuclein levels, for use in a method of treating a human subject suffering from, or being at risk of suffering from, a progressive brain disease, comprising providing a stimulator of LRP10, preferably a stimulator of LRPlO-mediated extracellular alpha-synuclein levels, and administering said stimulator of LRP10 to said human subject.

The invention further provides a use of a stimulator of LRP10, preferably a stimulator of LRPlO-mediated modulation of the ratio of intra/extra cellular alpha- synuclein levels, in the preparation of a medicament for treatment of a human subject suffering from, or being at risk of suffering from, a progressive brain disease, wherein the medicament comprising said stimulator of LRP10 is administered to said human subject.

The invention further provides a composition comprising a stimulator of LRP10 such as a viral vector expressing LRP10, and/or BAPTA AM, and a pharmaceutically acceptable excipient. Said pharmaceutically acceptable excipient preferably is selected from diluents, binders or granulating ingredients, a carbohydrate such as starch, a starch derivative such as starch acetate and/or maltodextrin, a polyol such as xylitol, sorbitol and/or mannitol, lactose such as ci- lactose monohydrate, anhydrous a-lactose, anhydrous 6-lactose, spray-dried lactose, and/or agglomerated lactose, sugars such as dextrose, maltose, dextrate and/or inulin, glidants (flow aids) and lubricants, and combinations thereof.

Said pharmaceutical composition for intracranial administration preferably is a sterile isotonic solution. Said buffer preferably is a citrate -based buffer, preferably lithium-, sodium-, potassium-, or calcium- citrate monohydrate, citrate trihydrate, citrate tetrahydrate, citrate pentahydrate, or citrate heptahydrate; lithium, sodium, potassium, or calcium lactate; lithium, sodium, potassium, or calcium phosphate; lithium, sodium, potassium, or calcium maleate; lithium, sodium, potassium, or calcium tartarate; lithium, sodium, potassium, or calcium succinate; or lithium, sodium, potassium, or calcium acetate, or a combination of two or more of the above. The pH of said buffer may be adjusted, preferably to a pH of 7.27 - 7.37 by hydrochloric acid, sodium hydroxide, citric acid, phosphoric acid, lactic acid, tartaric acid, succinic acid, or a combination of two or more of the above. The volume of the pharmaceutical composition that is administered may range from 0.5 ml to 5 ml. Said excipient preferably is selected from, but not limited to, urea, L- histidine, L- threonine, L-asparagine, L- serine, L-glutamine, polysorbate, polyethylene glycol, propylene glycol, polypropylene glycol, or a combination of two or more of the above.

A pharmaceutical composition as defined herein above may further comprise a non ergot dopamine agonist such as cabergoline, pramipexole and/or ropinerole. The administration of a pharmaceutical composition as defined herein above may be combined with oral administration of carbidopa ((2S)-3-(3,4-dihydroxyphenyl)-2- hydrazinyl-2-methylpropanoic acid), a dopa decarboxylase inhibitor, and L-DOPA ((2S) -2- amino- 3-(3, 4- dihydroxyphenyl)prop anoic acid), selegiline (2R)-N-methyl-l- phenyl-N-prop-2-ynylpropan-2-amine) and/or rasagiline ((lR)-N-prop-2-ynyl-2,3- dihydro- lH-inden- 1-amine).

Said pharmaceutical composition comprising a stimulator of LRP10 preferably is for use in a method of treatment of a progressive brain disease, preferably of Parkinson Disease (PD), Parkinson Disease Dementia (PDD) and Dementia with Lewy bodies (DLB). Said composition preferably is administered to a person in need thereof by local, intracranial administration by injection or by infusion. Said injection or infusion may be accomplished by use of external pump or of a fully implantable device. Said external pump is preferably equipped with a percutaneous catheter, tunneled or not tunneled, or equipped with a subcutaneous injection port and an implanted catheter. An implantable drug delivery system with a constant flow may be beneficial for long-term delivery of said composition.

A method of treating a human subject suffering from, or being at risk of suffering from, a progressive brain disease, especially from Parkinson Disease (PD), Parkinson Disease Dementia (PDD) and Dementia with Lewy bodies (DLB) may further comprise restoration of the altered sequence of a copy of the LRP10 encoding gene such that a wild type sequence is obtained, or such that a less devastating form of the LRPO protein is generated. Said restoration may be accomplished, for example, by CRISPR-CAS through gene editing (Sander and Joung, 2014. Nature Biotech 32, 347-355) or RNA editing (Abudayyeh et al., 2017. Nature 550: 280-284, 2017), and/or exon skipping (McNally et al., 2016. J Clin Invest 126: 1236-1238).

Examples

Example 1

Specimens

Clinically diagnosed patients with PD, PDD, or DLB (or the next of kin, for patients with dementia) and unaffected relatives provided written informed consent to the use of clinical data and biological samples for this study. From the pathologically diagnosed patients from the Netherlands Brain Bank, Amsterdam, and the Laboratory of Neuropathology, University of Bologna, written informed consent for brain autopsy and the use of clinical information and material for research purposes had been obtained from the donor or from the next of kin.

Study protocols were approved by the relevant Ethical Authorities. Clinical diagnosis of PD was made according to the UK PD Society Brain Bank criteria (Hughes ET AL., 1992. J Neurol Neurosurg Psychiatry 55: 181-4). PDD was diagnosed in patients developing dementia within one year from the onset of PD symptoms (McKeith et al., 2005. Neurology 65: 1863-72). DLB clinical diagnosis was based on the third report of the DLB consortium (McKeith et al., 2005. Ibid). The participants are described separately for the three study stages.

Stage I. Initial family. A large Italian family with PD segregating as an autosomal dominant trait was identified, neurologically examined, and biological specimens collected from 19 relatives (ten affected, nine unaffected; Family 1).

Stage II. International series of unrelated probands. A series of unrelated probands with PD, PDD, or DLB were ascertained between January 1st, 2000 and December 31st, 2017 at several centers from the International Parkinsonism Genetics Network, from the Netherlands Brain Bank (NBB), Netherlands Institute of Neuroscience, Amsterdam (www.brainbank.nl ), and from the Laboratory of Neuropathology, University of Bologna, Italy (LNUB). Whole Exome Sequencing (WES) data from subjects from a Dutch study of abdominal aortic aneurysms (AAA study) were included as controls; AAA study participants had provided written informed consent to the use of their WES data for genetic research. Data on neurological diseases are not available in the AAA study.

Stage III. Case-control series from three populations. Independent series of patients with clinically diagnosed PD and unaffected controls from Sardinia, Taiwan and Portugal were also studied. The controls were spouses of PD cases or unrelated individuals examined at the same centers, with no signs nor family history of PD, PDD and DLB.

Procedures

For genetic studies, DNA and RNA specimens were isolated from blood and brain samples following standard procedures. The Gentra Puregene Kit (Qiagen, Hilden, Germany) was used for isolation and purification of high-molecular weight genomic DNA from blood and brain specimens. DNA samples from 10 affected members of Family 1 underwent genome -wide SNP array genotyping with

HumanCNV370 bead chip (356,075 markers; Illumina, San Diego, CA, USA). . Analysis of copy number variants on the chrl4q locus was performed using Nexus Copy Number, Discovery Edition, v -8 (BioDiscovery, El Segundo, CA, USA).

Whole exome sequencing (WES) was performed in patient IV-2 of Family 1 using in-solution capturing (Illumina TruSeq kit), and an Illumina HiSeq2000 sequencer with 100 paired-end sequencing. The average coverage was >90X with 85% of the target regions covered >20X. Reads were aligned to the human reference genome build 19 (hgl9) using Burrows-Wheeler- Aligner (BWA-MEM) (Li and Durbin, 2009. Bioinformatics 25: 1754-60). Variant calling was done with GATK v3 (Genome-Analysis-Tool-Kit) (McKenna et al., 2010. Genome Res 20: 1297-303).

In Family 7, WES was performed using the Agilent Technologies (Santa Clara, CA, USA) SureSelect clinical relevant exons (CRE) capture kit, and an Illumina Hiseq2500 sequencer with paired-ends lOlbp + 9bp index (III- 1, III-4, and IV- 1), or an Illumina HiSeq4000 sequencer with paired-ends 150bp + 9bp index (Patient III-2). The average coverage was >90X, with 97% of the target regions covered more than 20X. The reads were aligned against the human reference genome build 19 (hgl9) using BWA-MEM. Subsequently, genetic variants were called using GATK v3 . Variants were filtered using Cartagenia Bench Lab NGS v -5.0.1 (Agilent Technologies). Within the genomic linkage region located on chromosome 14q variants were reported if they were i) heterozygous, ii) had a minor allele frequency (MAE) less than 0 -1% in dbSNP, Exome Variant Server NHLBI GO Exome Sequencing Project (ESP), 1000 Genomes

(http://www.internationalgenome.org/1000-genomes-browsers), Genome of the

Netherlands (GoNL), Exome Aggregation Consortium (ExAC), Genome aggregation database (GnomAD) iii) were exonic non-synonymous, iv) were within 5bp from a splice site. The splicing effect was evaluated in silico according to 5 splicing prediction programs (SpliceSite Finder-like, MaxEntScan, NNSPLICE,

GeneSplicer, and Human Splicing Finder) integrated in Alamut Visual version 4.2 (Interactive Biosoftware, Rouen, France). Variants were annotated using Annovar (Wang et al., 2010. Nucleic Acids Res 38: el64), and Mendelian clinically applicable pathogenicity score (M-CAP). The expression of genes harboring candidate variants according the afore-mentioned criteria was investigated using multiple datasets accessible in GeneCards® Human gene Database (http://www.genecards.org/) and in Unigene catalog (https://www.ncbi.nlm.nih.gov/unigene).

In the AAA study, WES was performed at Erasmus MC (Biomics), using an Illumina HiSeq2500 or HiSeq4000 sequencer. Of these samples, 464 were enriched using Agilents HaloPlex Target Enrichment System. The other 181 samples were enriched for clinically relevant exons using the Agilent Technologies SureSelect Clinical Research Exome (CRE) capture kit. Reads were aligned to the reference genome (hgl9) using BWA, and variants were called using the GATK software. Annotation was done using Annovar (Wang and Hakonarson, 2010. Ibid). The average whole exome coverage was >90 times, with 90% of the target regions covered more than 20 times. The average coverage of the LRP10 gene (coding exons, and intronic regions including 5 bp flanking the splicing sites) was 100 -7 times, of which 99% was covered more than 10 times. Variants were followed up if they had a MAF less than 0 -1% in ExAC, dbSNP, ESP, 1000 Genomes, GoNL, and GnomAD; if they were exonic non-synonymous, within 5bp from a splice site, and if they were predicted to be pathogenic with >5 in silico tools.

Using these criteria, only a single carrier of a LRP10 variant was identified among the entire group of 645 controls (1290 alleles). However, for our statistical analyses, a smaller number of alleles (n=1248) were considered, as a result of the following procedure:

553 subjects were unrelated probands in the AAA study, contributing therefore 1106 independent LRP10 alleles to our analysis;

50 subjects were close relatives of probands; our direct, systematic inspection of their WES data at the LRP10 locus revealed that they did not share any of their 2 haplotypes with their relative included in the 553 probands;

therefore, these 50 relatives contribute another 100 independent alleles to our analysis;

42 subjects were close relatives of other probands, and our inspection of their WES data at the LRP10 locus revealed that they shared only one haplotype with their relative included in the 553 probands; therefore, these 42 relatives contribute another 42 independent alleles to our analysis.

Sequence analysis on genomic DNA

Primers for the PCR amplification of LRP10 (all exons), LRRK2 (exons 29-48, coding for the functional LRRK2 protein domains, where the PD-causing mutations are located), SNCA (all exons), CHCHD2 (all exons), GBA (all exons), and VPS35 (fragment where the p.Asp620Asn pathogenic variant is located) are in Tables 1A, IB and 2.

Amplification reactions were performed in a total volume of 20 ml, containing lXFastStart Taq DNA Polymerase buffer, 200 mM of each dNTP, 0.5mM of forward primer, 0.5mM of reverse primer, 0.5 units of FastStart Taq DNA Polymerase (Roche, Basel, Switzerland), and 40ng of genomic DNA. Exon 1 was amplified with the addition of IX GC-RICH solution (Roche). PCR conditions: 5 minutes 94°C initial denaturation followed by 30 cycles of 30 seconds at 94°C; 30 seconds at 60°C; 90 seconds at 72° C, with a final extension for 5 minutes at 72°C. PCR reactions (4m1) were cleaned-up from unconsumed dNTPs and primers using 5 units of Exol and 0 -5 unit of Fast AP (Thermo Fisher Scientific, Waltham, MA, USA), 45 minutes at 37°C, 15 minutes at 80°C. Direct Sanger Sequencing was performed using Big Dye Terminator chemistry (version 3 -1; Thermo Fisher Scientific) as recommended by the manufacturer. Dye terminators were removed using SephadexG50 (GE Healthcare, Little Chalfont, UK) and loaded on an ABI 3730XL Genetic Analyzer (Thermo Fisher Scientific). Generated sequences were analyzed using the software packages Seqscape v3 -0 (Thermo Fisher Scientific) and Sequencing Analysis v60 (Thermo Fisher

Scientific).

LRP10 NM_014045.4, LRRK2 NM_198578.3, SNCA NM_000345.3, CHCHD2 NM_016139.3, GBA NM_000157.3, and VPS35 NM_018206 NCBI reference sequences were adopted for sequence variants annotation, and human genome variome society (HGVS) recommendations (den Dunnen et al., 2016. Hum Mutat 37: 564-9) were followed for variants nomenclature.

Multiple ligation probe amplification assay (MLPA, kit P051, MRC Holland, the Netherlands) was used to screen for copy number variants in the SNCA gene. High-resolution melting (HRM) analysis was adopted for studying LRP10 c.2095C>T (p.Pro699Ser) variant in an additional series of patients and controls from Sardinia; LRP10 c.l424+5delG variant in an independent series from Taiwan; and LRP10 e.l598G>T (p.Arg533Leu) variant in an independent series from

Portugal. In each experiment, positive and negative controls were included. The reaction was performed in a total volume of 11 ml, containing 5 ml SsoFastTM Eva Green® supermix (Bio-Rad, Hercules, CA, USA), 2mM primer forward and primer reverse, and 25 ng of genomic DNA. HRM condition: 3 minutes 98°C initial denaturation followed by 39 cycles of 5 seconds at 98°C; and 10 seconds at 58°C.

The melting curve was set between 70°C and 95°C. Carriers of variants identified during these experiments were confirmed by Sanger sequencing using the above- mentioned protocols. LRP10 variants that did not pass our filtering criteria mentioned above are reported in Table 6.

RNA isolation, cDNA preparation, and full length cDNA amplification The RNeasy procedure (RNeasy Mini Kit, Qiagen) was used to isolate and purify total RNA from different tissues as recommended by the manufacturer. cDNA synthesis was performed in the first step using total RNA (0.5 mg) primed with random primers using the Superscript® III First-Strand Synthesis System for RT-PCR (Thermo Fisher Scientific), followed by a RNase H digestion to remove the RNA template from the cDNAiRNA hybrid molecule. In the second step, full length cDNA LRP10 NM_014045.4 fragments were amplified in a total volume of 20 ml, containing IX GC Buffer II (5 mM Mg2+ plus), 400 mM of each dNTP, 0.5 mM of forward primer, 0.5 mM of reverse primer, 1 unit TaKaRa LA Taq (TAKARA Bio Inc., Kusatsu, Shiga, Japan) and 1 ml cDNA template. PCR conditions: 1 minutes 94°C initial denaturation followed by 30 cycles of 30 seconds at 94°C; 30 seconds at 60°C; 2 minutes at 72°C, with a final extension for 5 minutes at 72°C. Fragments were analyzed on a 1 .2 % agarose gel, and sized using a 1 Kb Plus DNA Ladder (Thermo Fisher Scientific).

Neuropat, hological characterization

Human brain tissue from Patient II- 1 Family 8 and Patient II- 1 Family 9 was obtained from The Netherlands Brain Bank (NBB), Netherlands Institute for Neuroscience, Amsterdam. Patient III- 1 Family 7 was obtained from the brain tissue collection at the Laboratory of Neuropathology, University of Bologna (LNUB). In both collection, a written informed consent for a brain autopsy and the use of the material, and clinical information for research purposes had been obtained from the donor or from next of kin.

At autopsy, tissue blocks of the three cases were obtained from 23 different regions including frontal, occipital, cingulate, parietal, temporal, pre- and postcentral cortex, hippocampus, amygdala, substantia nigra, pons, medulla oblongata, cervical cord (not available for Patient III-l Family 7), caudate, putamen, globus pallidus, insular and olfactory cortex, subthalamus and thalamus, cerebellum and meninges. In addition, tissue blocks from basal forebrain and hypothalamus were available from Patient III- 1 Family 7 donor.

Tissue was fixed in formalin, embedded in paraffin and cut into 8 pm sections. Staining of selected regions was performed using hematoxylin and eosin, Congo red, Galiyas silver stain, and immunohistochemistry against alpha- synuclein (clone KM51, 1:500, Monos an, Uden, the Netherlands), amyloid-beta (clone 6f/3d, 1:100, , Agilent Technologies, and clone 4G8, 1:5,000, BioLegend, San Diego, CA, USA) and hyperphosphorylated tau (clone AT8, 1:100, Fujirebio Europe N.V., Ghent, Belgium). For pathological staging of alpha-synuclein, amyloid-beta, neurofibrillary and neuritic plaque pathology, diagnostic criteria were used according to the BrainNet Europe (Alafuzoff et al., 2009. Acta Neuropathol 117:

635-52; Alafuzoff et al., 2008. Brain Pathol 18: 484-96) and the National Institute on Aging-Alzheimer's Association guidelines (Hyman et al., 2012. Alzheimers Dement 8: 1-13; Mirra et al., 1991. Neurology 41: 479-86; Thai et al., 2002.

Neurology 2002; 58(12): 1791-800). Transmission light microscopy images were taken using a Leica DM500 microscope (Leica Microsystems, Wetzlar, Germany) with a 63x oil lens (HC PL-APO, numerical aperture 1.40) and Leica Application Suite version 44 software (Leica Microsystems).

Cycloheximide chase experiments

HEK293 cell lines were expanded in growth medium (DMEM, BE12-604F/U1, Lonza, Basel Switzerland , 10% FCS,), and plated on 6 -well culture plates in growth medium. At 80% confluency, cells were transfected with C- terminal V5 -tagged LRP10 wildtype and variant expression constructs using GenejuiceA® transfection reagent (Merck, Darmstadt, Germany) according to manufactures’ specifications and incubated at 37°C/5% CO2. After 48 hrs, Cycloheximide (C7698-1G, Merck) was added to each well at 40pg/ml (final concentration), and cells were incubated at 37°C/5% CO2 for indicated time points. After treatment, cells were washed with PBS, and further processed for Western blotting analysis.

Western blot analysis

HEK293 cells were lysed in RIPA lysis buffer (150mM NaCl, 1.0 % IGEPAL® CA-630, 0.5% sodium deoxycholate, 0.1% SDS, and 50mM Tris, pH 8.0) containing protease inhibitor Complete® (Merck). Lysates were cleared by centrifugation at 10,000g for 10 min at 4°C. Protein concentrations were determined via Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific). For Western blotting, proteins were separated on 4-15% Criterion TGX precast gels (Bio-Rad), and transferred to nitrocellulose using the Trans-Blot® Turbo™ Transfer System (Bio-Rad). Blots were blocked using 5% blotting grade blocker nonfat milk (Bio-Rad) in PBS, 0.1% v/v TWEEN® 20 (Merck) for 30 minutes at room temperature. Primary antibody incubations were performed overnight at 4°C. Primary antibodies used: anti-V5- Tag (D3H8Q) rabbit monoclonal antibody (Cell Signaling Technologies, Danvers, MA, USA, 13202s, 1:2000 dilution); anti-Vinculin mouse monoclonal antibody (V284) (Santa Cruz Biotechnology, Dallas TX, USA, 1:2000 dilution). After washing in PBS, 0.1% v/v TWEEN® 20, blots were incubated for 1 hour at room

temperature with fluorescently conjugated Goat anti-mouse (IRDye 800) and goat

Table 1A. Primers for PCR amplification of the known PD genes

Table IB. Primers for PGR amplification of the known PD genes

anti-rabbit (IRDye 680) secondary antibodies (LLCOR Biosciences, Lincoln, NE, USA). After washing in PBS, 0.1% v/v TWEEN® 20, the blots were imaged and analyzed using an Odyssey Imaging system (LI- COR Biosciences),

Cell-surface labeling of live cells

HEK293 cell lines were expanded in growth medium (DMEM, BE12-604F/U1, Lonza, 10% ECS), and plated on glass coverslips placed in 12-well culture plates in growth medium. At 80% confluency, cells were transfected in duplicate with N- terminal V5 -tagged LRP10 wildtype and variant expression constructs using Genejuice® transfection reagent (Merck) according to manufactures’ specifications and incubated at 37°C/5% CO2. After 48 hrs, one set of the coverslip with transfected cells was used for cell-surface labelling (surface LRP10) and the other set of coverslips was used for labelling of permeabilized cells (total LRP10). The surface LRP10 cells were placed on an ice, and medium was replaced with ice-cold DMEM/HEPES (pH 7 -4, 25mM) for 5 minutes to block endocytosis. The total LRP10 cells remained in the incubator at 37°C/5% CO2. Subsequently, on the surface LRP10 cells, DMEM/HEPES was replaced with ice-cold DMEM/HEPES containing anti-V5-Tag (D3H8Q) rabbit monoclonal antibody (Cell Signaling Technologies 13202s, 1:500 dilution) and cells were incubated for 1 hour on ice to achieve cell- surface labeling of N-terminal V5-tagged LRP10. Next, after washing the coverslips in ice-cold PBS, cells on coverslips were fixed with 4% paraformaldehyde for 10 minutes. At this point the total LRP10 cells were also fixed with 4%

paraformaldehyde for 10 minutes. After washing in PBS, the surface LRP10 cells were incubated for 30 minutes with blocking buffer (2% [w/v] bovine serum albumin [BSA, A- 3294, Merck], 0.1M Glycine in PBS). The total LRP10 cells were incubated for 30 minutes with blocking buffer containing 0.1% Triton X-100 to permeabilize the cells (2% [w/v] BSA, 0.1M glycine, 0.1% [v/v] Triton X-100 in PBS). Secondary antibody incubation of surface LRP10 cells was performed overnight at 4°C in PBS containing 2% BSA, with the Alexa Fluor® 488 AffiniPure donkey anti-rabbit IgG antibody (Jackson ImmunoRese arch Laboratories, West Grove, PA, USA, 711-545-152, 1:500). Primary antibody incubation of Triton X-100 permeabilized total LRP10 cells was performed overnight at 4°C in PBS containing 2% BSA, with the anti-V5-Tag (D3H8Q) rabbit monoclonal antibody (Cell Signaling Technologies 13202s, 1:500 dilution). The next day, the surface LRP10 cells were washed 3 times in PBS, once in water, air dried and mounted in ProLong Gold with DAPI (Thermo Fisher Scientific, P-36931). Triton X-100 permeabilized total LRP10 cells were washed 3 times in PBS, and secondary antibody incubation of cell- surface labelled cells was performed for 1 hour at room temperature in PBS containing 2% BSA, with the Alexa Fluor® 488 AffiniPure donkey anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories, 711-545-152, 1:500). The above- mentioned PBS washing was repeated, followed by one wash in water, and subsequently the cells were air dried and mounted in ProLong Gold with DAPI (Thermo Fisher Scientific, P-36931). Staining was analyzed using a Leica SP5 confocal microscope (Leica Microsystems) and images were processed using Fiji software (Schindelin et al., 2012. Nat Methods 9: 676-82) keeping intensity levels constant for all images. Briefly, in Fiji software, cells of each condition were selected and outlines of the cells of interest were drawn using the bright field exposure of the field and the freeform drawing tool of Fiji software. Measurements were set from the analyze menu selecting: area, integrated density and mean gray value. Next, by selecting the‘measure' option from the analyze menu, area and intensity values of each cell were calculated, and transported to Excel software.

Background signal from areas surrounding the cells were selected for

normalization purposes. Corrected total cell fluorescence (CTCF) were calculated in Excel (CTCF = Integrated Density - [Area of selected cell X Mean fluorescence of background readings]). Cells were counted from 3 independent experiments with at least 40 cells counted per condition. Statistical analysis

For cycloheximide chase experiments, signal intensities of C-terminal V5- tagged LRP10 protein expression at each time point were normalized to Vinculin expression levels of the same time point. Subsequently, C-terminal V5-tagged LRP10 protein expression levels at time point 0 (untreated sample) were set at 100% and C-terminal V5-tagged LRP10 protein expression levels at all subsequent time points were expressed as percentage of untreated control for all analyzed C- terminal V5-tagged LRP10 wildtype and variant protein expression constructs. All experiments were repeated at least 4 times. Statistical significance levels were determined by Prism 7 software (Graphpad Software, Inc., Sand Diego, CA, USA). Column statistical analysis showed normal distribution of the data, and therefore a One-way ANOVA and Dunnett’s multiple comparisons test was adopted. Values passing the threshold for statistical significance of p<0 -05 were reported. For cell- surface labeling of live cells CTCF values of cell- surface N- terminal V5-tagged LRP10 wildtype and variant expression levels were counted from 3 independent experiments with at least 40 cells counted per condition. Mean CTCF values were calculated and Statistical significance levels were determined by Prism 7 software (Graphpad Software, Inc.). Column statistical analysis showed that the data were not normally-distributed, and therefore a Kruskal- Wallis and Dunn's multiple comparisons test was adopted. Values passing the threshold for statistical significance of p0.05 were reported.

Antibodies and LRP10 expression constructs

Primary antibodies: anti-V5-Tag (D3H8Q) rabbit monoclonal antibody (Cell Signaling Technologies 13202s,); anti- Vinculin mouse monoclonal antibody (V284) (Santa Cruz Biotechnology); alpha-synuclein (clone KM51, Monosan, Uden, the Netherlands); amyloid-beta (clone 6f)3d, Agilent Technologies);

hyperphosphorylated tau (clone AT8, Fujirebio Europe N.V., Ghent, Belgium); anti- TGN46 sheep polyclonal antibody (Bio-Rad, ahp500gt), anti-EEAl mouse monoclonal antibody (BD Biosciences, Los Angeles, CA, USA, clone 14, 610456); anti-GGAl mouse monoclonal (Santa Cruz Biotechnology, D-6, sc-271927), anti- VPS35 goat polyclonal antibody (Abeam, Cambridge, UK, ab 10099).

Secondary antibodies: goat anti-mouse IRDye 800CW (LI -COR Biosciences, 926-32210); goat anti-rabbit IRDye 680RD (LI-COR Biosciences, 926-32221); Alexa Fluor® 488 AffiniPure donkey anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories, 711-545-152); Alexa Fluor® 647-conjugated donkey anti-sheep (Jackson ImmunoResearch Laboratories); Alexa Fluor® 594-conjugated donkey anti-rabbit (Jackson ImmunoResearch Laboratories); Alexa Fluor® 488-conjugated donkey anti-mouse (Jackson Immuno Research Laboratories); Indodicarbocyanine, Cy5-conjugated donkey anti-mouse (Jackson Immunoresearch Laboratories); Alexa Fluor® 488-conjugated donkey anti-goat (Jackson ImmunoResearch Laboratories). Cloning of LRP10 expression constructs

The MGC Human LRP10 Sequence -Verified cDNA was subcloned from the human LRP 10-p CR4-TOPO plasmid (Dharmacon, Inc., Lafayette, CO, USA, Clone

ID: 8322768) into pcDNA™3 .1/V5-His-TOPO® using the pcDNA™3.1/V5-His TOPO® TA Expression Kit (Thermo Fisher Scientific, K480001) according to manufacturers’ specifications. This generated the C-terminal V5-tagged LRP10 wildtype expression construct. This construct was used to generate the N- terminal V5-tagged LRP10 wildtype expression construct. Briefly, via PCR-cloning the C- terminal V5 tag was removed and replaced by a translational stop signal following the last amino acid coding for full length LRP 10. Next, a BspEl site was introduced directly following the LRP10 signal peptide cleavage site (after amino acid 16). Sense and antisense oligonucleotides coding for the N-terminal V5-tag and a BspEl complementary overhang were generated (Integrated DNA Technologies, Inc.,

Coralville, I A, USA) and annealed. The annealed oligonucleotides were cloned into the BspEl digested transition construct, generating a N-terminal V5-tag in frame with the mature LRP 10 coding sequence resulting in the N-terminal V5-tagged LRP10 wildtype expression construct. Introduction of the p.Gly603Arg,

p.Pro699Ser, p.Arg533Leu, p.Tyr307Asn, p.Arg235Cys, p.Asn517del, variants in C- terminal V5-tagged and N-terminal V5-tagged LRP 10 wildtype expression constructs was achieved using the QuikChange Lightning Site-Directed

Mutagenesis Kit (Agilent Technologies, 210518) according to manufactures’ specifications. The N-terminal V5-tagged LRP 10 A2DXXLL mutant construct was generated via PCR-cloning, creating a translational stop signal following amino acid 689, removing the two DXXLL sorting motifs at the C-terminal tail of LRP10 (Boucher et al., 2008. Histochem Cell Biol 130: 315-27; Doray et al., 2008. Traffic 9: 1551-62). The two DXXLL sorting motifs located in LRP10 C-terminal are necessary for LRP10 internalization from the plasma membrane, and therefore the LRP10 A2DXXLL construct is used as an artificial positive control. All expression constructs were verified by Sanger sequencing.

Human iPSC culture and neural differentiation

The iPSC control line was provided by the Erasmus MC iPS core facility, and it has been characterized and described previously (V anhauwaert et al., 2017. EMBO J 36: 1392-411). Briefly, the iPSCs were grown on irradiated MEFs and cultured in normal iPSC medium (DMEM/F12 Advanced (Termo Fisher Scientific) supplemented with 20% KOSR, 2mM 1- glutamine, 0 -lmM MEM-NEAA, 0 -lmM 2- mercaptoethanol, 100U/ml penicillin/streptomycin (all fromThermo Fisher

Scientific) and 10ng/ml bFGF (Perprotech) at 37°C/5% CO2. Medium was changed daily and cells were passaged every 4-6 days either mechanically or enzymatically with collagenase type IV (100U/ml) (Thermo Fisher Scientific), and addition of 10mM ROCK inhibitor (Merck) to the medium after passaging.

Small molecule neural progenitor cells (smNPCs) were created and

differentiated into neurons according to published protocols (Reinhardt et al., 2013. EMBO J 36: 1392-411) with minor modifications. Briefly, iPSC colonies were detached from the MEFs 3-4 days after splitting, using 2 mg/mL collagenase IV. Pieces of colonies were collected by sedimentation and resuspended in iPSC medium (without FGF2) supplemented with 10 mM SB-431542 (Abeam), 1 mM dorsomorphin (Tocris Bioscience, Bristol, UK) for neural induction, as well as 3 mM CHIR 99021 (Axon MedChem, Groningen, the Netherlands) and 0.5 mM PMA (Enzo Biochem. Inc., Farmingdale, NY, USA), and cultured in Petri dishes.

Medium was replaced on day 2 by N2B27 medium supplemented with the same small molecule supplements. N2B27 medium consisted of DMEM-F12

(Invitrogen)/Neurobasal (Thermo Fisher Scientific) 50:50 with 1:200 N2

supplement (Thermo Fisher Scientific), 1:100 B27 supplement lacking vitamin A (Thermo Fisher Scientific) with 1% penicillin/streptomycin/glutamine (PAA). On day 4, SB-431542 and dorsomorphin were withdrawn and 150 mM ascorbic acid (AA;Merck) was added to the medium. On day 6, the embryoid bodies (EBs), which showed intensive neuroepithelial outgrowth, were triturated with a 1,000 mL pipette into smaller pieces and plated on Matrigel-coated (Matrigel, growth factor reduced, high concentration; BD Biosciences) 12-well plates at a density of about 1C)— 15 pieces per well in smNPC expansion medium (N2B27 with CHIR, PMA, and AA). The first split was performed at a 1:5 to 1:10 ratio on days 2 to 4 after plating. All the remaining splitting ratios were at least 1:10. After passage 4, PMA was switched to 0.5 mM smoothened agonist (SAG). After a maximum of 5 splits, cultures were virtually free of contaminating non-smNPCs.

To start differentiation into neurons, a homogeneous suspension of 50,000 to 100,000 smNPCs were seeded onto PDL and matrigel-coated coverslips in 150 mΐ droplets of patterning medium (N2B27 with lng/ml GDNF, 2ng/ml BDNF, 200 mM AA, 0.5 mM SAG). Patterning medium was changed every other day. After six to eight days in patterning medium, the medium was changed to maturation medium (N2B27 with 2 ng/ml GDNF, 2 ng/ml BDNF, 1 ng/ml TGF-b3, 200 mM AA, 100 mM dbcAMP). The first time when the maturation medium was added, 5 ng/ml activinA was included. All other changes of maturation medium, 2ng/ml activinA was included. Maturation medium was exchanged every third day. Mature neurons were processed for imaging 12 weeks after the start of differentiation.

Transfection and Immunocytochemistry in neurons

Differentiated neurons were transfected two times on subsequent days with N-terminal V5-tagged LRP10 wildtype expression plasmid using DNA-In® neuro transfection reagent according to manufacturer’s specifications (Thermo Fisher Scientific). After 4 days, Cells were fixed for 20 minutes, and washed with PBS. Subsequently, primary antibody incubation was performed overnight at 4°C in labeling buffer (50 mM Tris.Cl [pH 7 -4], 0.9% NaCl, 0.25% gelatin, 0.2% Triton X100 [all from Merck]). The next day coverslips were washed in PBS, followed by secondary antibody incubation in the dark for 1 hour at room temperature in labeling buffer. After washing in PBS, coverslips were mounted with Prolong Gold plus DAPI (Thermo Fisher Scientific). Primary antibodies: anti-V5-tag (D3H8Q) rabbit monoclonal antibody (Cell Signaling Technologies 13202s, 1:500 dilution), sheep anti-TGN46 (Bio-Rad, ahp500gt, 1:200), mouse anti-EEAl (BD Biosciences, [clone 14], 610456, 1:200), mouse anti-GGAl (Santa Cruz Biotechnology, [D-6], sc- 271927, 1:100), goat anti-VPS35 (Abcam, ab 10099, 1:200). Secondary antibodies:

Alexa Fluor® 647-conjugate d donkey anti-sheep (Jackson); Alexa Fluor® 594- conjugate d donkey anti-rabbit (Jackson ImmunoResearch Laboratories), Alexa Fluor® 488-conjugate d donkey anti-mouse (Jackson ImmunoResearch Laboratories) indodicarbocyanine, Cy5 -conjugated donkey anti-mouse (Jackson ImmunoResearch Laboratories); Alexa Fluor® 488-conjugated donkey anti-goat. Slides were analyzed on a Leica SP5 AOBS confocal laser scanning microscope with a HCX PL APO CS x40 objective (NA 1.25) or HCX PL APO CS x63 objective (NA 1.40). Image stacks were acquired with a step size of 494nm over 9.88 mm thickness and a resolution of 1024x1024 pixels. Images were processed using Fiji software.

Stage I. After excluding mutations in the genes causing autosomal dominant PD: SNCA, LRRK2, VPS35, and CHCHD2, as well as GBA variants, as is indicated herein above, a genome -wide SNP- array genotyping was performed in ten affected relatives from Family 1, and run parametric multipoint linkage analysis assuming an autosomal dominant mode of inheritance. Whole exome sequencing (WES) was performed in the index patient (IV-2, Fig. 2). Variants were annotated with

Annovar (Wang and Hakonarson, 2010. Nucleic Acids Res 38: el64) and M-CAP (Jagadeesh et al, 2016. Nat Genet 48: 1581-6). The variants located within the linkage interval were then filtered using the following criteria: (1) heterozygous state; (2) rarity, defined as MAF<0.1% in Exome Aggregation Consortium (ExAC; http ://exac.broadinstitute .org/)), dbSNP

(https://www.ncbi.nlm.nih.gov/projects/SNP/), Exome Variant Server NHLBI GO Exome Sequencing Project (ESP (http://evs.gs.washington.edu/EVS/)), Genome of the Netherlands (GoNL (www.nlgenome.nl)), and the genome aggregation database (GnomAD (http://gnomad.broadinstitute.org/)); (3) exonic and non-synonymous, or, predicted to affect splicing in silico; (4) pathogenicity, defined as so predicted by at least five of eleven in silico tools, as is indicated herein above. This work led to the nomination of LRP10 (low-density lipoprotein receptor related protein 10) as the candidate disease-causing gene in family 1 (See Figure 2). Sanger sequencing was used for validation and co-segregation analysis in all 19 members of this family.

Stage II. Subsequently, the entire LRP10 open reading frame (ORF) and exon-intron boundaries were sequenced in 660 unrelated probands with PD, PDD, or DLB (Sanger sequencing in 659, WES in one) (primers in Table 2). Variants fulfilling the same criteria mentioned in stage I were considered of interest. Sanger sequencing was used for co-segregation analysis when DNA from additional relatives was available. Furthermore, we searched for LRP10 variants (entire coding region and exon-intron boundaries) in the WES database of the individuals from the AAA study (average LRP10 depth coverage 100E7 times). Variants fulfilling the same, above-specified criteria, were considered, and their frequency compared to that in our series of 660 patients with PD, PDD, and DLB.

Stage III. By high-resolution melting analysis (HRM) we studied three of the identified LRP10 variants in independent population-matched series of patients and controls from Sardinia, Taiwan and Portugal (Table 1). The genes causing autosomal dominant PD: SNCA, LRRK2, VPS35, and CHCHD2, as well as the risk gene GBA, were also analyzed in the proband of all families where LRP10 variants were identified.

For pathological analysis, autopsy tissue blocks were obtained from 23 different brain regions as indicated herein above, fixed in formalin, embedded in paraffin and cut into 8 pm sections. Staining of selected regions was performed using hematoxylin and eosin, Congo red, Galiyas silver stain, and

immunohistochemistry (details of antibodies and diagnostic criteria for

pathological staging are provided herein above).

The effect of the identified LRP10 potentially pathogenic variants on the stability of LRP10 protein and subcellular localization was studied. Human induced neurons were derived from previously-characterized induced pluripotent stem cells (iPSC)20 according to published protocols (Reinhardt et al., 2013. PLoS One 8: e59252), with minor modifications, as is detailed herein above.

Differentiated neurons were transfected with N-terminal V5-tagged LRP10 wildtype expression plasmid, and further processed for immunoeytochemistry. Statistical Analysis

Continuous clinical variables (e.g. age at disease onset, age at examination, disease duration) are described as means and standard deviations (SD). We performed parametric multipoint linkage analysis with the software MERLIN (version 1.1.2) (Abecasis et al., 2002. Nat Genet 30: 97-101) with affected-only analysis assuming an autosomal dominant model of disease inheritance, equal markers allele frequency, disease allele frequency 1x10⁰⁵, and penetrance 0.002 (wild type) / 0.99 (heterozygous carrier) / 0.99 (homozygous carrier). LOD scores >3.3 were considered genome-wide significant. Categorical data were compared using Fisher’s Exact test. A two-sided p<0.05 was considered significant. For stability and subcellular localization of LRP10 protein, statistical analyses were performed with Prism 7 software (Graphpad Software, Inc.).

Bole of the funding source

The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.

Results

Stage I. In Family 1 (see Fig. 2), 14 individuals suffered from PD, and 10 of them were alive and personally examined; mean age at disease onset was 59.8 years (SD 17 ±8.7; range 46-73) (Additional clinical features are provided in Table 3). A single genome-wide significant linkage peak (LOD 3.301) was identified on chromosome 14pl3-ql2 (data not shown). Copy number aberrations within the linkage interval were not detected by SNP-array analysis using Nexus. In the WES analysis of the index case, only three heterozygous variants, in the OR11H12, POTEG and LRP10 genes, located within the linkage region, had MAF<0 .1% (data not shown). The OR11H12 and POTEG variants are predicted as benign by the majority of in silico tools and have a negative nucleotide conservation score

(Genomic Evolutionary Rate Profiling (GERP) = -0.993 and -0.546, respectively) (Table 4). Instead, the LRP10 variant, c 1807G>A (p.Gly603Arg, NM_014045.4), is predicted to be damaging by 7/11 tools (Table 4), has a high GERP score (+4.9), and replaces a very conserved residue in the LRP10 protein. Furthermore, LRP10 is the only of these three genes with evidence of expression in human brain. LRP10 was then nominated as the candidate disease-causing gene in Family 1. We confirmed by Sanger sequencing the presence of the LRP10 c 1807G>A variant in the index patient (data not shown), and all nine affected, as well as three unaffected relatives (III- 14, IV-3, and IV- 6; age at last examination: 58, 49, and 45 years). The other four unaffected relatives did not carry the LRP10 variant (Fig. 2).

Stage II. Next, we Sanger-sequenced all LRP10 exons and exon-intron boundaries in 660 unrelated probands, including 430 clinically diagnosed familial PD (n=420) or PDD (n=10), 62 clinically diagnosed DEB, and 168 pathologically confirmed PD (n=49), PDD (n=74) or DEB (n=45). A total of eight variants were identified that fulfilled our criteria (heterozygous, rare, exonic and non- synonymous, or predicted to affect splicing, and predicted as pathogenic by >5 tools; Tables 4 and 5). These variants are described below.

Among 430 probands with familial PD or PDD, we detected c.2095C>T (p.Pro699Ser) in a Sardinian PD proband, and confirmed it in his affected PD cousin (Family 2). A second variant, the deletion of a guanine at position +5 in LRP10 intron 5 (c 1424+5delG) was identified in a Taiwanese PD proband (Family 3), and confirmed in two PD relatives. A third variant, c.1598G>T (p.Arg533Leu), was identified in a Portuguese PD proband and in her PD sibling (Family 4). Last, in an Italian PDD proband (Family 5) a fourth variant was identified (c.919T>A p.Tyr307Asn). Among 62 clinically diagnosed DLB, in a Dutch proband affected with probable DLB, we detected a G/A substitution at position +5 in LRP10 intron 5 (c.!424+5G>A, Family 6). Very interestingly, the c.!424+5G>A variant replaces the same guanine deleted by the Taiwanese variant c 1424+5delG.

In the brain DNA samples from our 168 pathologically confirmed probands, three LRP10 variants were detected, and characterized as germline by confirming their presence in DNA from blood samples of the same individuals. We identified c.703C>T p.Arg235Cys in an Italian proband with familial PDD (Family 7). She was initially diagnosed with PD and later developed rapidly progressive dementia. Three affected relatives developed a similar neurodegenerative illness, including parkinsonism, dementia, and also additional neurological signs, such as

supranuclear gaze palsy and ideomotor apraxia (data not shown). The p.Arg235Cys variant was also present in two of these affected relatives. The remaining relative displayed a similar disease (including supranuclear gaze limitation) but, interestingly, he had a much longer disease course (~20 years before death) compared to only 8-14 years in the relatives who carried the LRP10 variant, and might represent a phenocopy. Another variant (an in-frame deletion of three nucleotides, c.l549_1551delAAT), was identified in a Dutch DLB proband (Family 8). This deletion removes the asparagine at position 517 in the LRP10 protein (p.Asn517del). Last, a frameshift variant (c.632dupT, p . Ala212Serfs* 17), was detected in a Dutch PD proband with severe parkinsonism and mild cognitive Table 2. PCR primers for LRP10 genomic regions and cDNA

impairment, and confirmed in her sister with dementia, but in none of four unaffected siblings (Family 9). An overview of the identified LRP10 pathogenic variants identified in PD, PDD and DLB patients is provided in Table 5. Among 645 Dutch elderly controls from the AAA study, only one carrier of a LRP10 variant was identified, that fulfilled our criteria (c.451C>T, p.Argl51Cys) (neurological status of this subject not available). The frequency of LRP10 variants, defined according to the above mentioned criteria, in our probands of European ancestry (seven carriers in 608 probands excluding Brazilian and Taiwanese patients) is significantly higher than that in the AAA series (one carrier) (two-sided Fisher’s exact test, p=0.0306).

Stage III. In the independent series of unrelated PD patients and unrelated controls (412 PD, 242 controls Sardinia; 831 PD, 431 controls from Taiwan; and 223 PD, 138 controls from Portugal; Table 1), the c.2095C>T (p.Pro699Ser) variant was detected in two of 412 Sardinian cases and none of 242 controls; one case had familial PD (Family 10) and the variant was also found in the affected sib; the other had sporadic PD (Family 11). The c.l424+5delG variant was found in one of 831 Taiwanese PD probands (a familial PD, Family 12) and none of 431 controls; the variant was also present in one affected sibling. Last, the c 1598G>T

(p.Arg533Leu) was absent in our case -control series from Portugal (223 patients, 138 controls).

The analyses of the known PD-causing genes, SNCA, LRRK2, VPS35, and CHCHD2, as well as the risk gene GBA, in the eleven probands carrying the above- mentioned LRP10 variants, revealed no causative mutations (by either Sanger sequencing or MLPA). Only one GBA risk variant, c.508C>T (p.Argl70Cys, common nomenclature p.Argl31Cys), was present in the proband of Family 11. The average age at symptoms onset in all the LRP10 variant carriers considered together (PD, PDD and DLB, n=30) was 62.6 years (SD 9.2, range 46-75).

Pathological examination

Macroscopic examination of the brain of the three above-mentioned patients with LRP10 variants showed a pale substantia nigra and locus coeruleus, with only mild atrophy limited to the parietal regions (Patient II- 1 Family 8) an isolated small amygdala (Patient II- 1 family 9), and moderate atrophy in the amygdala and hippocampus (Patient III-l Family 7). Microscopy showed severe loss of

neuromelanin-containing neurons in the substantia nigra (data not shown), and many LBs and LNs throughout the brain, compatible with the highest Braak alpha-synuclein stage (stage 6; data not shown). The dorsal motor nucleus of the vagal nerve, locus coeruleus and substantia nigra showed classical brainstem-type LBs (data not shown). In addition, crescent-shaped and annular alpha-synuclein- 1 immunore active neuronal inclusions were found in the CAS region and especially in the amygdala (data not shown), similar to those previously described in PD cases with pathogenic SNCA variants (Pasanen et al., 2014. Neurobiol Aging 35: 2180 el- 5; Kiely et al., 2015. Neurodegener 10: 41). In patient II- 1, Family 9 (data not shown), many alpha -synuclein-positive glial inclusions were present in the substantia nigra and putamen. Alzheimer’s disease (AD) pathology (Montine et al., 2012. Acta Neuropathol 123: 1-11) was of intermediate grade in one (Patient II- 1 Family 8), and mild in the other two brains (Patient II- 1 Family 9, Patient III- 1 Family 7).

The two splicing variants (c 1424delG identified in two Taiwanese PD families, and C.1424G>A in one Dutch DLB family) are predicted to affect mRNA splicing by all in silico tools (data not shown). We therefore evaluated mRNA splicing in five patients carrying the c.l424+5delG variant (Taiwanese Families 3 and 12), and the proband carrying the C.1424+5G>A variant (Dutch Family 6). LRP10 cDNA amplification revealed an identical, aberrant pattern in all patients carrying either substitution (Fig. 3).

By sequencing the aberrant cDNA species, we show the preferential usage of a different upstream splice donor site in exon 5 (position c.517_c.518) resulting in the incorporation of a much shorter, aberrant exon 5 which misses 907 coding nucleotides (r.518 to r.1424), and at protein level, a frameshift with premature truncation and removal of a large part of the LRP10 protein (p . Gly 173 Alafs* 34) . Sanger sequencing of the LRP10 cDNA in the patient carrying the c.632dupT (p .Ala212Serfs* 17) variant (Patient II- 1 Family 9) only yielded the wild type transcript (data not shown), suggesting nonsense-mediated mRNA decay. This is supported by reduced mRNA levels in brain tissue of the donor with

p.Ala212Serfs*17 compared to brain tissue from unaffected elderly subjects and donors with idiopathic PD and DLB (data not shown). mRNA expression and stability was normal in the patient carrying the c.1549_1551delAAT (p.Asn517del) variant (II- 1 from Family 8; data not shown). Table 3. Clinical features in patients carrying LRP10 pathogenic variants

In our functional studies, we show that the p.Tyr307Asn, p.Gly603Arg, p.Arg235Cys, and p.Pro699Ser variants significantly reduce LRPlO protein stability as compared to wildtype LRP10 (Fig. 4A, 4B). Interestingly, variants that did not affect LRP10 protein stability (p.Asn517del, and p.Arg533Leu) display significantly increased surface labeling in vitro in cells overexpressing LRP10 (Fig. 5), indicating that in these variants LRPlO subcellular localization is markedly affected. Furthermore, in 12 week-old human neuronal cultures, V5-tagged LRP10 localized to vesicular structures in the neuronal soma and neurites (data not shown). Interestingly, both in the cell soma and neurites, V5-tagged LRP10 strongly co-localized with the TGN marker TGN46 and GGA1 (Golgi Associated, Gamma Adaptin Ear Containing, ARE Binding Protein 1), (data not shown).

Furthermore, in the soma, V5-tagged LRP10 also partially co-localized with the early endosomal marker EEA1 and retromer marker VPS 35 (data not shown). These data show that in neurons LRP10 is localized to vesicular structures, including endosomes, retromer, and trans-Golgi network (TGN).

Conclusion

Here we report nine rare LRP10 variants associated with familial PD, PDD and DLB, and provide initial evidence for a role of the LRP10 protein in the pathogenesis of the neurodegenerative disorders with LB pathology. By studying a large multi-incident PD kindred, we nominate LRP10 as the candidate disease causing gene. Subsequently, we found eleven probands, who each carried one of eight LRP10 potentially pathogenic variants, and the same variants were detected in nine out of the ten relatives available for testing, providing independent, albeit limited, evidence of co-segregation with disease.

A further remarkable observation is that the same guanine at position c.1424+5 in LRP10 intron 5 was the target of two different alterations (a deletion and a substitution), rare or absent in control databases, and detected in patients with PD or DLB from three families and two distant populations. These two variants result in an identical, severe aberration at the level of mRNA splicing. Although small copy-number variants within the linkage region, and non-coding variants or repeat expansions could have been missed because whole genome sequencing was not performed, taken as a whole, our genetic data provide compelling evidence that LRP10 variants are implicated in the development of neurodegenerative diseases ranging from PD and PDD to DLB.

The identification of LRP10 variants in some unaffected family members suggests that the penetrance of at least some of these variants is age-related, and perhaps incomplete. Three p.Gly603Arg carriers in Family 1 were free from symptoms and clinical signs at age 58 (III- 5), 49 (IV- 3) and 45 (IV-6), and four p.Pro699Ser carriers did not manifest disease symptoms or signs at age 87 and 76 (III-2 and III-5 Family 2), 70 (II -3 Family 10) and 67 (II-4 Family 10), suggesting a lower penetrance compared to the other LRP10 variants. This would be in line with the available evidence of reduced penetrance for variants in SNCA18

(Papadimitriou et al., 2016. Mov Disord 31: 1226-30) and especially LRRK2 (Goldwurm et al., 2011. Mov Disord 26: 2144-5). Follow-up studies of these and other LRP10 families to be identified in the future might lead to accurate penetrance estimates. Furthermore, how LRP10 variants relate to phenotypes varying from typical PD to PDD, or DLB, remains currently unknown. Other genetic or non- genetic modifiers are possibly involved and further studies are warranted.

An abundant load of alpha- synuclein aggregation in the form of brainstem and cortical LBs and LNs was present in all the three brains studied here, with different LRP10 variants (p.Arg235Cys, p.Asn517del, and p.Ala212Serfs*), suggesting an important involvement of LRP10 in the molecular cascades leading to alpha-synucleinopathy.

Example 2

HEK293T cells transfected with alpha-synuclein protein expression construct together with a GFP protein expression construct show high intracellular alpha-synuclein protein expression levels, but low extracellular alpha-synuclein protein expression levels (Figure 6, lane indicated by the left arrow). On the contrary, the same alpha-synuclein expression construct transfected in the same cell type together with LRP10 expression construct leads to high intracellular expression of alpha-synuclein protein, but also high extracellular expression levels of alpha-synuclein protein (Figure 6. lane indicated by arrow on the right). These data indicate that LRP 10 can specifically regulate the extracellular levels of alpha-synuclein.

Furthermore, we have been able to show that intra-extracellular

partitioning of alpha-synuclein mediated by LRP 10 activity can be modulated after addition of small molecules (Figure 7). In a preliminary small scale screen we have been able to show that BAPTA AM can increase the LRP10 mediated extracellular levels of alpha-synuclein, whereas Ionomycin does not affect the LRP10 mediated extracellular levels of alpha- synuclein. One of skill in the art will understand that other cell permeable Ca2+ chelators may be used in aspects of te present invention in alternative embodiments. Based on our findings, we conclude that LRP10 mutations in PD and DLB patients can interfere with the clearance of alpha-synuclein in the brain, leading to alpha-synucleinopathy. These data support LRP10 as a potential drug target to regulate alpha-synuclein processing in PD.

Claims

Claims

1. A method of typing a human subject as suffering from, or being at risk of suffering from, an inherited progressive brain disease selected from Parkinson Disease (PD), Parkinson Disease Dementia (PDD) and Dementia with Lewy bodies (DLB), the method comprising:

a) providing a sample comprising cells, or parts thereof, of said subject;

b) determining in said cells or cell parts an amount and/or a level of activity of low density lipoprotein receptor-related protein 10 (LRP10) or the presence of one or more mutations in the LRP10 gene that affect stability or subcellular localization of the LRP10 protein;

c) correlating said determined amount and/or level of activity, or the presence of said one or more mutations, to a healthy control; and

d) typing the human subject as suffering from, or being at risk of suffering from, said inherited progressive brain disease if the determined amount and/or level of activity of LRP10 is reduced, when compared to the healthy control, or if said one or more mutations in the LRP10 gene is present.

2. The method according to claim 1, wherein a level of activity of low density lipoprotein receptor-related protein 10 (LRP10) is determined by sequence analysis of a LRP10-encoding gene, and wherein a reduced amount and/or a level of activity of the protein is indicated when said sequence analysis indicates the presence of one or more mutations in the LRP10 gene encoding pathogenic variants of the LRP10 protein with reduced stability or altered subcellular localization.

3. The method according to any one of claims 1-2, wherein a level of activity of low density lipoprotein receptor-related protein 10 (LRP10) is determined by sequence analysis of one or more of human genomic coordinates 23346401, 23346689, 23345586, 23346192, 23345076, 23344860, 23346022, 23346023, 23346024 and 23344789 of NC_000014.8.

4. The method according to claim 1 or 2, comprising determining an amount and/or a level of protein activity of LRP10 in said cells or cell parts.

5. The method of any one of claims 1 or 4, wherein an amount and/or level of protein activity of LRP10 is determined in cellular vesicles, preferably in exosomes.

6. The method of claim 5, wherein the exosomes are isolated from a bodily fluid, preferably selected from blood, saliva, tears, urine and cerebrospinal fluid.

7. A method of screening for a level of activity of low density lipoprotein receptor-related protein 10 (LRP10), comprising:

providing a cell that expresses LRP10, and optionally alpha-synuclein, and determining a level of activity of LRP10, optionally by determining the amount or ratio of intra/extracellular levels of alpha-synuclein.

8. A method of screening for a candidate compound for treating progressive brain disease, comprising:

providing a cell that expresses LRP10,

adding one or more compounds to said cell, and

determining a level of LRP10 activity in said cell, preferably by determining the amount or ratio of intra/extra cellular levels of alpha-synuclein, and

designating said one or more compounds as candidate compound(s) for treating progressive brain disease, referred to herein as stimulator of LRP10, in case said LRP10 activity is increased.

9. The method of claim 8, comprising identifying a compound that modulates the ratio of intra/extra cellular levels of alpha-synuclein.

10. The method of any one of claims 7-9, wherein the cell expresses a mutated LRP10 protein, preferably comprising one or more mutations at human genomic coordinates 23346401, 23346689, 23345586, 23346192, 23345076, 23344860, 23346022, 23346023, 23346024 and 23344789 of NC_000014.8.

11. A method of treating a human subject suffering from, or being at risk of suffering from, a progressive brain disease, comprising:

providing a stimulator of LRP10, preferably a stimulator of LRPlO-mediated modulation of the ratio of intra/extracellular levels of alpha-synuclein, and administering said stimulator of LRP10 to said human subject.

12. The method of claim 11, wherein the stimulator of LRP10 is an expression construct that expresses low density lipoprotein receptor-related protein 10 (LRP10) or a protein that is at least 70% identical to LRP10 and/or BAPTA AM.

13. The method according to claim 11 or 12, wherein the stimulator of LRP10 is provided to the brain.

14. A composition comprising a stimulator of LRP10 and a pharmaceutically acceptable excipient.