Clinical, neuroimaging and genetic findings in Brazilian patients with neurodegeneration with brain iron accumulation.

Neurodegeneration with brain iron accumulation (NBIA) encompasses a clinically and genetically heterogeneous group of rare disorders. Here, we report clinical, neuroimaging and genetic studies in twenty three Brazilian NBIA patients. In thirteen subjects, deleterious variants were detected in known NBIA-causing genes (PANK2, PLA2G6, C9ORF12, WDR45 and FA2H), including previously unreported variants in PANK2 and PLA2G6. Two patients carried rare, likely pathogenic variants in genes not previously associated with NBIA: KMT2A c.11785A > C (p.Ile3929Leu), and TIMM8A c.127T > C (p.Cys43Arg), suggesting an expansion of their associated phenotypes to include NBIA. In eight patients the etiology remains unsolved, suggesting variants undetectable by the adopted methods, or the existence of additional NBIA-causing genes.

LRP10 and α-synuclein transmission in Lewy body diseases.

Autosomal dominant variants in LRP10 have been identified in patients with Lewy body diseases (LBDs), including Parkinson's disease (PD), Parkinson's disease-dementia (PDD), and dementia with Lewy bodies (DLB). Nevertheless, there is little mechanistic insight into the role of LRP10 in disease pathogenesis. In the brains of control individuals, LRP10 is typically expressed in non-neuronal cells like astrocytes and neurovasculature, but in idiopathic and genetic cases of PD, PDD, and DLB, it is also present in α-synuclein-positive neuronal Lewy bodies. These observations raise the questions of what leads to the accumulation of LRP10 in Lewy bodies and whether a possible interaction between LRP10 and α-synuclein plays a role in disease pathogenesis. Here, we demonstrate that wild-type LRP10 is secreted via extracellular vesicles (EVs) and can be internalised via clathrin-dependent endocytosis. Additionally, we show that LRP10 secretion is highly sensitive to autophagy inhibition, which induces the formation of atypical LRP10 vesicular structures in neurons in human-induced pluripotent stem cells (iPSC)-derived brain organoids. Furthermore, we show that LRP10 overexpression leads to a strong induction of monomeric α-synuclein secretion, together with time-dependent, stress-sensitive changes in intracellular α-synuclein levels. Interestingly, patient-derived astrocytes carrying the c.1424 + 5G > A LRP10 variant secrete aberrant high-molecular-weight species of LRP10 in EV-free media fractions. Finally, we show that this truncated patient-derived LRP10 protein species (LRP10splice) binds to wild-type LRP10, reduces LRP10 wild-type levels, and antagonises the effect of LRP10 on α-synuclein levels and distribution. Together, this work provides initial evidence for a possible functional role of LRP10 in LBDs by modulating intra- and extracellular α-synuclein levels, and pathogenic mechanisms linked to the disease-associated c.1424 + 5G > A LRP10 variant, pointing towards potentially important disease mechanisms in LBDs.

deCLUTTER2+ - a pipeline to analyze calcium traces in a stem cell model for ventral midbrain patterned astrocytes.

Astrocytes are the most populous cell type of the human central nervous system and are essential for physiological brain function. Increasing evidence suggests multiple roles for astrocytes in Parkinson's disease, nudging a shift in the research focus, which historically pivoted around ventral midbrain dopaminergic neurons (vmDANs). Studying human astrocytes and other cell types in vivo remains challenging. However, in vitro-reprogrammed human stem cell-based models provide a promising alternative. Here, we describe a novel protocol for astrocyte differentiation from human stem cell-derived vmDAN-generating progenitors. This protocol simulates the regionalization, gliogenic switch, radial migration and final differentiation that occur in the developing human brain. We characterized the morphological, molecular and functional features of these ventral midbrain patterned astrocytes with a broad palette of techniques and identified novel candidate midbrain-astrocyte specific markers. In addition, we developed a new pipeline for calcium imaging data analysis called deCLUTTER2+ (deconvolution of Ca2+ fluorescent patterns) that can be used to discover spontaneous or cue-dependent patterns of Ca2+ transients. Altogether, our protocol enables the characterization of the functional properties of human ventral midbrain patterned astrocytes under physiological conditions and in disease.

PTPA variants and impaired PP2A activity in early-onset parkinsonism with intellectual disability.

The protein phosphatase 2A complex (PP2A), the major Ser/Thr phosphatase in the brain, is involved in a number of signalling pathways and functions, including the regulation of crucial proteins for neurodegeneration, such as alpha-synuclein, tau and LRRK2. Here, we report the identification of variants in the PTPA/PPP2R4 gene, encoding a major PP2A activator, in two families with early-onset parkinsonism and intellectual disability. We carried out clinical studies and genetic analyses, including genome-wide linkage analysis, whole-exome sequencing, and Sanger sequencing of candidate variants. We next performed functional studies on the disease-associated variants in cultured cells and knock-down of ptpa in Drosophila melanogaster. We first identified a homozygous PTPA variant, c.893T>G (p.Met298Arg), in patients from a South African family with early-onset parkinsonism and intellectual disability. Screening of a large series of additional families yielded a second homozygous variant, c.512C>A (p.Ala171Asp), in a Libyan family with a similar phenotype. Both variants co-segregate with disease in the respective families. The affected subjects display juvenile-onset parkinsonism and intellectual disability. The motor symptoms were responsive to treatment with levodopa and deep brain stimulation of the subthalamic nucleus. In overexpression studies, both the PTPA p.Ala171Asp and p.Met298Arg variants were associated with decreased PTPA RNA stability and decreased PTPA protein levels; the p.Ala171Asp variant additionally displayed decreased PTPA protein stability. Crucially, expression of both variants was associated with decreased PP2A complex levels and impaired PP2A phosphatase activation. PTPA orthologue knock-down in Drosophila neurons induced a significant impairment of locomotion in the climbing test. This defect was age-dependent and fully reversed by L-DOPA treatment. We conclude that bi-allelic missense PTPA variants associated with impaired activation of the PP2A phosphatase cause autosomal recessive early-onset parkinsonism with intellectual disability. Our findings might also provide new insights for understanding the role of the PP2A complex in the pathogenesis of more common forms of neurodegeneration.

A new alpha-synuclein missense variant (Thr72Met) in two Turkish families with Parkinson's disease.

INTRODUCTION: Missense variants and multiplications of the alpha-synuclein gene (SNCA) are established as rare causes of autosomal dominant forms of Parkinson's Disease (PD). METHODS: Two families of Turkish origins with PD were studied; the SNCA coding region was analyzed by Sanger sequencing, and by whole exome sequencing (WES) in the index patient of the first and the second family, respectively. Co-segregation studies and haplotype analysis across the SNCA locus were carried out. Functional studies included in vitro thioflavin-T aggregation assay and in silico structural modelling of the alpha-synuclein (α-syn) protein. RESULTS: We identified a novel heterozygous SNCA variant, c.215C > T (p.Thr72Met), segregating with PD in a total of four members in the two families. A shared haplotype across the SNCA locus was found among variant carriers, suggestive of a common ancestor. We next showed that the Thr72Met α-syn displays enhanced aggregation in-vitro, compared to the wild-type species. In silico analysis of a tetrameric α-syn structural model revealed that Threonine 72 lies in the tetrameric interface, and substitution with the much larger methionine residue could potentially destabilize the tetramer. CONCLUSION: We present clinical, genetic, and functional data supporting a causative role of the SNCA c.215C > T (p.Thr72Met) variant in familial PD. Testing for this variant in patients with PD, especially of Turkish origin, might detect additional carriers. Further functional analyses might offer new insights into the shared biochemical properties of the PD-causing SNCA missense variants, and how they lead to neurodegeneration.

LRP10 interacts with SORL1 in the intracellular vesicle trafficking pathway in non-neuronal brain cells and localises to Lewy bodies in Parkinson's disease and dementia with Lewy bodies.

Loss-of-function variants in the low-density lipoprotein receptor-related protein 10 (LRP10) gene have been associated with autosomal-dominant Parkinson's disease (PD), PD dementia, and dementia with Lewy bodies (DLB). Moreover, LRP10 variants have been found in individuals diagnosed with progressive supranuclear palsy and amyotrophic lateral sclerosis. Despite this genetic evidence, little is known about the expression and function of LRP10 protein in the human brain under physiological or pathological conditions. To better understand how LRP10 variants lead to neurodegeneration, we first performed an in-depth characterisation of LRP10 expression in post-mortem brains and human-induced pluripotent stem cell (iPSC)-derived astrocytes and neurons from control subjects. In adult human brain, LRP10 is mainly expressed in astrocytes and neurovasculature but undetectable in neurons. Similarly, LRP10 is highly expressed in iPSC-derived astrocytes but cannot be observed in iPSC-derived neurons. In astrocytes, LRP10 is present at trans-Golgi network, plasma membrane, retromer, and early endosomes. Interestingly, LRP10 also partially co-localises and interacts with sortilin-related receptor 1 (SORL1). Furthermore, although LRP10 expression and localisation in the substantia nigra of most idiopathic PD and DLB patients and LRP10 variant carriers diagnosed with PD or DLB appeared unchanged compared to control subjects, significantly enlarged LRP10-positive vesicles were detected in a patient carrying the LRP10 p.Arg235Cys variant. Last, LRP10 was detected in Lewy bodies (LB) at late maturation stages in brains from idiopathic PD and DLB patients and in LRP10 variant carriers. In conclusion, high LRP10 expression in non-neuronal cells and undetectable levels in neurons of control subjects indicate that LRP10-mediated pathogenicity is initiated via cell non-autonomous mechanisms, potentially involving the interaction of LRP10 with SORL1 in vesicle trafficking pathways. Together with the specific pattern of LRP10 incorporation into mature LBs, these data support an important mechanistic role for disturbed vesicle trafficking and loss of LRP10 function in neurodegenerative diseases.

EIF2AK2 Missense Variants Associated with Early Onset Generalized Dystonia.

OBJECTIVE: The study was undertaken to identify a monogenic cause of early onset, generalized dystonia. METHODS: Methods consisted of genome-wide linkage analysis, exome and Sanger sequencing, clinical neurological examination, brain magnetic resonance imaging, and protein expression studies in skin fibroblasts from patients. RESULTS: We identified a heterozygous variant, c.388G>A, p.Gly130Arg, in the eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2) gene, segregating with early onset isolated generalized dystonia in 5 patients of a Taiwanese family. EIF2AK2 sequencing in 191 unrelated patients with unexplained dystonia yielded 2 unrelated Caucasian patients with an identical heterozygous c.388G>A, p.Gly130Arg variant, occurring de novo in one case, another patient carrying a different heterozygous variant, c.413G>C, p.Gly138Ala, and one last patient, born from consanguineous parents, carrying a third, homozygous variant c.95A>C, p.Asn32Thr. These 3 missense variants are absent from gnomAD, and are located in functional domains of the encoded protein. In 3 patients, additional neurological manifestations were present, including intellectual disability and spasticity. EIF2AK2 encodes a kinase (protein kinase R [PKR]) that phosphorylates eukaryotic translation initiation factor 2 alpha (eIF2α), which orchestrates the cellular stress response. Our expression studies showed abnormally enhanced activation of the cellular stress response, monitored by PKR-mediated phosphorylation of eIF2α, in fibroblasts from patients with EIF2AK2 variants. Intriguingly, PKR can also be regulated by PRKRA (protein interferon-inducible double-stranded RNA-dependent protein kinase activator A), the product of another gene causing monogenic dystonia. INTERPRETATION: We identified EIF2AK2 variants implicated in early onset generalized dystonia, which can be dominantly or recessively inherited, or occur de novo. Our findings provide direct evidence for a key role of a dysfunctional eIF2α pathway in the pathogenesis of dystonia. ANN NEUROL 2021;89:485-497.

Low prevalence of known pathogenic mutations in dominant PD genes: A Swedish multicenter study.

OBJECTIVE: To determine the frequency of mutations known to cause autosomal dominant Parkinson disease (PD) in a series with more than 10% of Sweden's estimated number of PD patients. METHODS: The Swedish Parkinson Disease Genetics Network was formed as a national multicenter consortium of clinical researchers who together have access to DNA from a total of 2,206 PD patients; 85.4% were from population-based studies. Samples were analyzed centrally for known pathogenic mutations in SNCA (duplications/triplications, p.Ala30Pro, p.Ala53Thr) and LRRK2 (p.Asn1437His, p.Arg1441His, p.Tyr1699Cys, p.Gly2019Ser, p.Ile2020Thr). We compared the frequency of these mutations in Swedish patients with published PD series and the gnomAD database. RESULTS: A family history of PD in first- and/or second-degree relatives was reported by 21.6% of participants. Twelve patients (0.54%) carried LRRK2 p.(Gly2019Ser) mutations, one patient (0.045%) an SNCA duplication. The frequency of LRRK2 p.(Gly2019Ser) carriers was 0.11% in a matched Swedish control cohort and a similar 0.098% in total gnomAD, but there was a marked difference between ethnicities in gnomAD, with 42-fold higher frequency among Ashkenazi Jews than all others combined. CONCLUSIONS: In relative terms, the LRRK2 p.(Gly2019Ser) variant is the most frequent mutation among Swedish or international PD patients, and in gnomAD. SNCA duplications were the second most common of the mutations examined. In absolute terms, however, these known pathogenic variants in dominant PD genes are generally very rare and can only explain a minute fraction of familial aggregation of PD. Additional genetic and environmental mechanisms may explain the frequent co-occurrence of PD in close relatives.

Late-onset phenotype associated with a homozygous GJC2 missense mutation in a Turkish family.

OBJECTIVE: Recessive mutations in the Gap Junction Protein Gamma 2 (GJC2) gene cause Pelizaeus-Merzbacher-like disease type 1, a severe infantile-onset hypomyelinating leukodystrophy. Milder, late-onset phenotypes including complicated spastic paraplegia in one family (SPG44), and mild tremor in one case, were reported associated to GJC2 homozygous missense mutations. Here, we report a new family with two siblings carrying a different homozygous GJC2 mutation, presenting with late-onset ataxic and pyramidal disturbances, and parkinsonism in one of them. METHODS: Two affected siblings were studied by neurological examination and brain MRI. Genetic analyses included genome-wide homozygosity mapping in both siblings, and whole exome sequencing in one sib. The resulting candidate gene variant was validated by Sanger sequencing. RESULTS: The affected siblings share a novel homozygous GJC2 missense mutation (c.820G>C, p.Val274Leu), predicted as pathogenic by all used in-silico tools. Brain MRI showed hyperintense signal in T2-weighted images in the internal capsule and subcortical and periventricular white matter, consistent with hypomyelination. CONCLUSIONS: Our findings confirm and further expand the late-onset phenotypes of GJC2 mutations, to include prominent ataxia, pyramidal disturbances and mild parkinsonism, and confirm the distinctive associated MRI pattern.

LRP10 variants in Parkinson's disease and dementia with Lewy bodies in the South-West of the Netherlands.

OBJECTIVE: To analyse LRP10 variants, recently associated with the development of Parkinson's disease (PD), Parkinson's disease dementia (PDD) and dementia with Lewy bodies (DLB), in a series of patients and controls from the South-West of the Netherlands (Walcheren). METHODS: A series of 130 patients with PD, PDD or DLB were clinically examined, and a structured questionnaire used to collect information about family history of PD and dementia. The entire LRP10 coding region was sequenced by Sanger methods in all patients, and haplotype analysis was performed for one recurrent LRP10 variant. The fragments containing possibly pathogenic LRP10 variants were sequenced in 62 unaffected control subjects from the same region. Other known PD-associated genes were analyzed by exome sequencing and gene dosage in the carriers of LRP10 variants. RESULTS: Four patients were carriers of a rare heterozygous, possibly pathogenic LRP10 variant: p.Arg151Cys, p.Arg263His, and p.Tyr307Asn. None of these variants was detected among the controls, nor were additional mutations identified in known PD-associated genes in the four LRP10 variant carriers. The previously reported p.Tyr307Asn variant was identified in two patients (with PD and PDD), who are connected genealogically within six generations, and in one of their relatives with cognitive decline. Haplotype analysis suggests a common founder for the p.Tyr307Asn variant carriers analyzed. DISCUSSION: We report three possibly pathogenic LRP10 variants in patients with PD and PDD from a local Dutch population. The identification of additional patients carrying the p.Tyr307Asn variant provides some further evidence that this variant is pathogenic for PD and PDD.

Candidate CSPG4 mutations and induced pluripotent stem cell modeling implicate oligodendrocyte progenitor cell dysfunction in familial schizophrenia.

Schizophrenia is highly heritable, yet its underlying pathophysiology remains largely unknown. Among the most well-replicated findings in neurobiological studies of schizophrenia are deficits in myelination and white matter integrity; however, direct etiological genetic and cellular evidence has thus far been lacking. Here, we implement a family-based approach for genetic discovery in schizophrenia combined with functional analysis using induced pluripotent stem cells (iPSCs). We observed familial segregation of two rare missense mutations in Chondroitin Sulfate Proteoglycan 4 (CSPG4) (c.391G > A [p.A131T], MAF 7.79 × 10-5 and c.2702T > G [p.V901G], MAF 2.51 × 10-3). The CSPG4A131T mutation was absent from the Swedish Schizophrenia Exome Sequencing Study (2536 cases, 2543 controls), while the CSPG4V901G mutation was nominally enriched in cases (11 cases vs. 3 controls, P = 0.026, OR 3.77, 95% CI 1.05-13.52). CSPG4/NG2 is a hallmark protein of oligodendrocyte progenitor cells (OPCs). iPSC-derived OPCs from CSPG4A131T mutation carriers exhibited abnormal post-translational processing (P = 0.029), subcellular localization of mutant NG2 (P = 0.007), as well as aberrant cellular morphology (P = 3.0 × 10-8), viability (P = 8.9 × 10-7), and myelination potential (P = 0.038). Moreover, transfection of healthy non-carrier sibling OPCs confirmed a pathogenic effect on cell survival of both the CSPG4A131T (P = 0.006) and CSPG4V901G (P = 3.4 × 10-4) mutations. Finally, in vivo diffusion tensor imaging of CSPG4A131T mutation carriers demonstrated a reduction of brain white matter integrity compared to unaffected sibling and matched general population controls (P = 2.2 × 10-5). Together, our findings provide a convergence of genetic and functional evidence to implicate OPC dysfunction as a candidate pathophysiological mechanism of familial schizophrenia.

PTRHD1 Loss-of-function mutation in an african family with juvenile-onset Parkinsonism and intellectual disability.

BACKGROUND: The genetic bases of PD in sub-Saharan African (SSA) populations remain poorly characterized, and analysis of SSA families with PD might lead to the discovery of novel disease-related genes. OBJECTIVES: To investigate the clinical features and identify the disease-causing gene in a black South African family with 3 members affected by juvenile-onset parkinsonism and intellectual disability. METHODS: Clinical evaluation, neuroimaging studies, whole-exome sequencing, homozygosity mapping, two-point linkage analysis, and Sanger sequencing of candidate variants. RESULT: A homozygous 28-nucleotide frameshift deletion in the PTRHD1 coding region was identified in the 3 affected family members and linked to the disease with genome-wide significant evidence. PTRHD1 was recently nominated as the disease-causing gene in two Iranian families, each containing 2 siblings with similar phenotypes and homozygous missense mutations. CONCLUSION: Together with the previous reports, we provide conclusive evidence that loss-of-function mutations in PTRHD1 cause autosomal-recessive juvenile parkinsonism and intellectual disability. © 2018 International Parkinson and Movement Disorder Society.

LRP10 genetic variants in familial Parkinson's disease and dementia with Lewy bodies: a genome-wide linkage and sequencing study.

BACKGROUND: Most patients with Parkinson's disease, Parkinson's disease dementia, and dementia with Lewy bodies do not carry mutations in known disease-causing genes. The aim of this study was to identify a novel gene implicated in the development of these disorders. METHODS: Our study was done in three stages. First, we did genome-wide linkage analysis of an Italian family with dominantly inherited Parkinson's disease to identify the disease locus. Second, we sequenced the candidate gene in an international multicentre series of unrelated probands who were diagnosed either clinically or pathologically with Parkinson's disease, Parkinson's disease dementia, or dementia with Lewy bodies. As a control, we used gene sequencing data from individuals with abdominal aortic aneurysms (who were not examined neurologically). Third, we enrolled an independent series of patients diagnosed clinically with Parkinson's disease and controls with no signs or family history of Parkinson's disease, Parkinson's disease dementia, or dementia with Lewy bodies from centres in Portugal, Sardinia, and Taiwan, and screened them for specific variants. We also did mRNA and brain pathology studies in three patients from the international multicentre series carrying disease-associated variants, and we did functional protein studies in in-vitro models, including neurons from induced pluripotent stem-like cells. FINDINGS: Molecular studies were done between Jan 1, 2008, and Dec 31, 2017. In the initial kindred of ten affected Italian individuals (mean age of disease onset 59·8 years [SD 8·7]), we detected significant linkage of Parkinson's disease to chromosome 14 and nominated LRP10 as the disease-causing gene. Among the international series of 660 probands, we identified eight individuals (four with Parkinson's disease, two with Parkinson's disease dementia, and two with dementia with Lewy bodies) who carried different, rare, potentially pathogenic LRP10 variants; one carrier was found among 645 controls with abdominal aortic aneurysms. In the independent series, two of these eight variants were detected in three additional Parkinson's disease probands (two from Sardinia and one from Taiwan) but in none of the controls. Of the 11 probands from the international and independent cohorts with LRP10 variants, ten had a positive family history of disease and DNA was available from ten affected relatives (in seven of these families). The LRP10 variants were present in nine of these ten relatives, providing independent-albeit limited-evidence of co-segregation with disease. Post-mortem studies in three patients carrying distinct LRP10 variants showed severe Lewy body pathology. Of nine variants identified in total (one in the initial family and eight in stage 2), three severely affected LRP10 expression and mRNA stability (1424+5delG, 1424+5G→A, and Ala212Serfs*17, shown by cDNA analysis), four affected protein stability (Tyr307Asn, Gly603Arg, Arg235Cys, and Pro699Ser, shown by cycloheximide-chase experiments), and two affected protein localisation (Asn517del and Arg533Leu; shown by immunocytochemistry), pointing to loss of LRP10 function as a common pathogenic mechanism. INTERPRETATION: Our findings implicate LRP10 gene defects in the development of inherited forms of α-synucleinopathies. Future elucidation of the function of the LRP10 protein and pathways could offer novel insights into mechanisms, biomarkers, and therapeutic targets. FUNDING: Stichting ParkinsonFonds, Dorpmans-Wigmans Stichting, Erasmus Medical Center, ZonMw-Memorabel programme, EU Joint Programme Neurodegenerative Disease Research (JPND), Parkinson's UK, Avtal om Läkarutbildning och Forskning (ALF) and Parkinsonfonden (Sweden), Lijf and Leven foundation, and cross-border grant of Alzheimer Netherlands-Ligue Européene Contre la Maladie d'Alzheimer (LECMA).

ACO2 homozygous missense mutation associated with complicated hereditary spastic paraplegia.

OBJECTIVE: To identify the clinical characteristics and genetic etiology of a family affected with hereditary spastic paraplegia (HSP). METHODS: Clinical, genetic, and functional analyses involving genome-wide linkage coupled to whole-exome sequencing in a consanguineous family with complicated HSP. RESULTS: A homozygous missense mutation was identified in the ACO2 gene (c.1240T>G p.Phe414Val) that segregated with HSP complicated by intellectual disability and microcephaly. Lymphoblastoid cell lines of homozygous carrier patients revealed significantly decreased activity of the mitochondrial aconitase enzyme and defective mitochondrial respiration. ACO2 encodes mitochondrial aconitase, an essential enzyme in the Krebs cycle. Recessive mutations in this gene have been previously associated with cerebellar ataxia. CONCLUSIONS: Our findings nominate ACO2 as a disease-causing gene for autosomal recessive complicated HSP and provide further support for the central role of mitochondrial defects in the pathogenesis of HSP.