Patient FL2-1 (Table 1 and Fig. 2) developed a mild depression followed shortly by forgetfulness at the age of 50 years. Two years later he had a flat affect, inappropriate behavior, poor concentration, executive dysfunction, restless legs syndrome, and insomnia. Examination 3 years after the onset of symptoms demonstrated psychomotor slowing, and ideomotor and constructional apraxia. The Mini-Mental State Examination (MMSE) score was 22/30. His gait was slow and shuffling. His postural stability was poor leading to frequent falls. He had rigidity and bradykinesia in all four extremities symmetrically. (a-b) Axial T2-weighted MR images showed localized hyperintense foci in both frontal and parietal lobes (long arrows), involving the periventricular, deep and subcortical white matter, sparing the subcortical U-fibers. Hyperintense focus in the right forceps minor (arrowhead) was seen. In the final stage of his illness, he became mute, reached a vegetative state, and died at the age of 55 years. Autopsy was performed. (c) Myelin loss in frontal white matter with a pigmented macrophage and a pale vacuolated axonal spheroid (Luxol fast blue). (d) Spheroids with phosphorylated neurofilament immunohistochemistry. (e) Spheroids with amyloid precursor protein immunohistochemistry. (f) Pigmented macrophages and reactive astrocytes (H&E). (g) White matter macrophages with HLA-DR immunohistochemistry. (h) Bizarre white matter astrocytes. (i) Ballooned cortical neurons with alpha-B-crystallin immunohistochemistry. Bar (c-i) = 30 μm

Abbreviated pedigrees of all families with HDLS included in this study. Filled symbols indicate affected individuals. An arrowhead indicates the proband. To protect confidentiality some individuals are not shown and sex is portrayed using a diamond for all individuals except affected individuals and their spouse. In each family, at least one affected family member received an autopsy (red pound sign) or biopsy (red star) confirmation of HDLS. A ‘+’ sign indicates that DNA was included in the CSFR1 sequencing analyses to confirm that mutations segregated with disease. For each patient with DNA available for genetic studies, a unique patient number (UPN) corresponding to Table 1 is included above the patient.

(a) The CSF1R gene extends over 60kb and contains 22 exons (vertical hatches). (b) Exon structure of the human CSF1R cDNA. Positions of the start codon (ATG) and stop codon (TGA) are indicated. For mutations detected, arrows indicate positions relative to exons and protein domains. (c) Domain structure of the CSF1R protein showing the immunoglobulin domains (IG) and the protein tyrosine kinase domain (PTK), interrupted by the kinase insert (shaded). (d) Fifteen heterozygote CSF1R mutations detected in 14 families with autopsy or biopsy proven HDLS and in one patient clinically diagnosed with corticobasal syndrome. Family identifiers, cDNA numbering (relative to NM_005211.3) and predicted translational changes are indicated. (e) ClustalW alignment for the parts of the PTK domain where the mutations occur, including multiple CSF1R homologs and all human CSF1/PDGF receptor family members. Comparison of human CSF1R (NP_005202.2), mouse CSF1R (NP_001032948.2), chicken CSF1R (XP_414597.2), Zebrafish CSF1R (NP_571747.1), human KIT (NP_000213.1), human FLT3 (NP_004110.2), human PDGFRα (NP_006197.1) and human PDGFRβ (NP002600.1). Amino acid positions of the mutations are indicated above the alignment.

Autophosphorylation of several tyrosine residues within the kinase domain of CSF1R is critical for its subsequent signaling involved in cell survival and proliferation. We studied CSF1R autophosphorylation in HeLa cells which do not express detectable levels of CSF-1 thereby minimizing endogenous CSF1-induced signaling. A representative Western blot of lysates from CSF1R_WT or mutant CSF1R_M875T transfected HeLa cells treated with CSF-1 for 5, 15, or 30 minutes is shown. Lysates from untreated CSF1R-transfected cells are included as a control and GAPDH immunoreactivity is shown to ensure equal protein loading. Total CSF1R immunodetection for both DNA constructs was robust. Further, we observed strong phosphorylation of wild-type CSF1R after 5 minutes of CSF-1 treatment, which decreased over the course of 15 and 30 minutes, as determined by immunoblotting using CSF1R phospho-specific tyrosine (p-Y) antibodies. In contrast, no CSF1R autophosphorylation at any of the selected tyrosine residues was detected after CSF-1 treatment in CSF1RM875T transfected cells. Experiments were repeated three times with similar outcome. Comparable results were obtained using CSF1R mutants CSF1RE633K and CSF1RM766T (Supplementary Fig. 5).