Familial encephalopathy with neuroserpin inclusion bodies

Abstract

We report on a new familial neurodegenerative disease with associated dementia that has presented clinically in the fifth decade, in both genders, and in each of several generations of a large family from New York State-a pattern of inheritance consistent with an autosomal dominant mode of transmission. A key pathological finding is the presence of neuronal inclusion bodies distributed throughout the gray matter of the cerebral cortex and in certain subcortical nuclei. These inclusions are distinct from any described previously and henceforth are identified as Collins bodies. The Collins bodies can be isolated by simple biochemical procedures and have a surprisingly simple composition; neuroserpin (a serine protease inhibitor) is their predominant component. An affinity-purified antibody against neuroserpin specifically labels the Collins bodies, confirming their chemical composition. Therefore, we propose a new disease entity-familial encephalopathy with neuroserpin inclusion bodies (FENIB). The conclusion that FENIB is a previously unrecognized neurodegenerative disease is supported by finding Collins bodies in a small kindred from Oregon with familial dementia who are unrelated to the New York family. The autosomal dominant inheritance strongly suggests that FENIB is caused by mutations in the neuroserpin gene, resulting in intracellular accumulation of the mutant protein.

Figure 1.

Histopathology of cerebral cortex and substantia nigra of an affected 57-year-old male (A–F) and an affected 60-year-old female (G and H). A: Low magnification shows microvacuolation and gliosis of layer II of cortex and several neuronal inclusions (arrows) in the deeper cortex (H&E staining; original magnification, ×40). B: Several inclusion bodies (arrows) in the deeper layers of frontal cortex (H&E staining; original magnification, ×200). C: Higher magnification (H&E staining; original magnification, ×400) shows inclusions within neuronal cytoplasm, compressing the remnant of a neuronal cell body and nucleus, and lying apparently free in the neuropil. D: Sections of substantia nigra pars compacta showing eosinophilic cytoplasmic inclusions of pigmented neurons superficially resembling Lewy bodies (H&E staining; original magnification, ×400). E: Unlike Lewy bodies, the inclusions are strongly PAS positive. Multiple inclusions affecting a single neuron are commonly seen (PAS staining; original magnification, ×400). F: Also in contrast to most Lewy bodies, the inclusions are nonreactive with ubiquitin immunohistochemistry (ubiquitin; original magnification, ×400). G and H: Fluorescein-labeled lectins applied to unfixed frozen sections of cerebral cortex and viewed by fluorescence microscopy (original magnification, ×250); G: Concanavalin A (specificity α- mannose, α- glucose); H: Lycopersicon esculentum (specificity (glc NAc)₃).

Figure 2.

Togographic distribution of Collins bodies. Coronal sections of the cerebral hemispheres: frontal (A), frontotemporal (B and C), and parieto-occipital (D). Transverse sections: midbrain (E), pons and cerebellum (F and G), and medulla oblongata and cerebellum (H). The composite figure summarizes data obtained from four autopsy cases of FENIB. (Templates redrawn and modified with permission from the authors of ref. 52 ).

Figure 3.

Transmission electron micrographs of inclusion bodies in cerebral cortex. A: Three inclusions in the cytoplasm of a cortical neuron. The nucleus shows only slight compression (original magnification, ×4000). B: Higher magnification of another inclusion body. Note amorphous, finely granular composition and apparent limiting membrane (original magnification, ×20,000).

Figure 4.

Isolation of inclusion bodies. The inclusions were isolated from 2 g of frozen cerebral cortical tissue obtained at autopsy from an affected individual. The presence of the inclusion bodies was monitored throughout the isolation by light microscopy after alcohol fixation and H&E staining. Complete details of the isolation are given in Materials and Methods.

Figure 5.

Visualization of inclusions by histochemistry and immunohistochemistry. The various fractions obtained during the isolation scheme were assayed for the presence of the inclusions by H&E staining (original magnification, ×400) (A–C). A: Starting material (unfixed, frozen brain tissue). B: Fraction labeled P₁ in Figure 4▶ . C: Fraction identified as P₅ in Figure 4▶ . The presence of neuroserpin was detected with a neuroserpin-specific polyclonal antibody (D–F; original magnification, ×400). D: Cortical tissue from an age- and gender-matched control; E: Cortical tissue from an affected individual; F: Fraction labeled P₅ in Figure 4▶ . Tissue from an affected individual from the Oregon family. G: Cortical tissue stained with H&E (original magnification, ×400). H: Cortical tissue stained with neuroserpin antibody (original magnification, ×400).

Figure 6.

SDS-PAGE. Fractions obtained from the isolation procedure were visualized by Coomassie blue staining after SDS-PAGE in a 7.5% gel. Lane 1: Homogenate. Lane 2: Fraction S₆ obtained after heating P₅ in SDS for 5 minutes at 75°C. Lane 3: Fraction S₆ obtained after heating P₅ in SDS for 2 hours at 75°C. Lane 4: Fraction S₆ obtained from age- and gender-matched control tissue. (Fraction S₆ was obtained by resuspending the Collins body-enriched fraction (P₅) in a volume of 4% SDS equal to 1/20 the original homogenization volume, heating for the various times as noted, and centrifuging for 5 minutes. The supernatant (50 μl) was applied to the gel.

Figure 7.

Identification of 57-kd protein. The 57-kd protein isolated from inclusion bodies was identified by its amino acid sequence. The N-terminal and internal peptide compositions of the protein were determined, and the resulting sequence information was analyzed by the BLAST program. The primary structure of human neuroserpin is presented in the top line. The first 16 amino acids are underlined and reportedly represent the signal sequence. The alignment of the peptides identified for the 57-kd protein is presented in bold letters below the known sequence. The only difference found between the wild-type neuroserpin and the 57-kd protein is a change of an aspartate for an asparagine at position 100 in the wild-type.

Figure 8.

Pedigree. The familial occurrence of the disease. Individuals were classified as affected by one of three categories: family history (); clinically (); or neuropathologically (⋄). Unaffected individuals are indicated by open symbols (⋄). The pedigree has been modified to protect the family’s privacy. Deceased individuals are not indicated. Roman numerals refer to the generation, Arabic numbers to individuals within that generation.