The dementia familial encephalopathy with neuroserpin inclusion bodies (FENIB) is caused by point mutations in the neuroserpin gene. We have shown a correlation between the predicted effect of the mutation and the number of intracerebral inclusions, and an inverse relationship with the age of onset of disease. Our previous work has shown that the intraneuronal inclusions in FENIB result from the sequential interaction between the reactive centre loop of one neuroserpin molecule with b-sheet A of the next. We show here that neuroserpin Portland (Ser52Arg), which causes a severe form of FENIB, also forms loop-sheet polymers but at a faster rate, in keeping with the more severe clinical phenotype. The Portland mutant has a normal unfolding transition in urea and a normal melting temperature but is inactive as a proteinase inhibitor. This results in part from the reactive loop being in a less accessible conformation to bind to the target enzyme, tissue plasminogen activator. These results, with those of the CD analysis, are in keeping with the reactive centre loop of neuroserpin Portland being partially inserted into b-sheet A to adopt a conformation similar to an intermediate on the polymerization pathway. Our data provide an explanation for the number of inclusions and the severity of dementia in FENIB associated with neuroserpin Portland. Moreover the inactivity of the mutant may result in uncontrolled activity of tissue plasminogen activator, and so explain the epileptic seizures seen in individuals with more severe forms of the disease.Keywords: conformational diseases; neuroserpin; polymerization; serpin; serpinopathies.The autosomal dominant dementia familial encephalopathy with neuroserpin inclusion bodies (FENIB) results from point mutations in the neuroserpin gene and is characterized by inclusions of neuroserpin within cortical and subcortical neurons [1][2][3]. Neuroserpin is a member of the serine proteinase inhibitor or serpin superfamily. It inhibits the enzyme tissue plasminogen activator (tPA) and may be important in regulating neuronal plasticity and memory [4][5][6][7]. We have recently expressed, purified, and characterized wild-type neuroserpin and neuroserpin with the Ser49Pro mutation, which was identified in the first reported family with FENIB [6]. The mutation reduced the inhibitory activity of neuroserpin by 100-fold and increased the formation of polymeric protein under physiological conditions. Neuroserpin polymers result from the sequential insertion of the reactive centre loop of one molecule into b-sheet A of another [1,6]. The resulting species is inactive as a proteinase inhibitor and accumulates in the endoplasmic reticulum in cell models of disease [8] and in vivo [2].Three other mutants of neuroserpin are now recognized to cause FENIB: Ser52Arg, His338Arg and Gly392Glu [3]. This condition is unusual among neurodegenerative disorders in that there is a striking correlation between the number of inclusions within the cerebral cortex and an inverse relationship with the age of onset...

Alzheimer disease is characterized by extracellular plaques composed of A␤ peptides. We show here that these plaques also contain the serine protease inhibitor neuroserpin and that neuroserpin forms a 1:1 binary complex with the N-terminal or middle parts of the A␤ 1-42 peptide. This complex inactivates neuroserpin as an inhibitor of tissue plasminogen activator and blocks the loop-sheet polymerization process that is characteristic of members of the serpin superfamily. In contrast neuroserpin accelerates the aggregation of A␤ 1-42 with the resulting species having an appearance that is distinct from the mature amyloid fibril. Neuroserpin reduces the cytotoxicity of A␤ 1-42 when assessed using standard cell assays, and the interaction has been confirmed in vivo in novel Drosophila models of disease. Taken together, these data show that neuroserpin interacts with A␤ 1-42 to form off-pathway non-toxic oligomers and so protects neurons in Alzheimer disease.Alzheimer disease is the most common form of dementia. The pathological features are characterized by neurofibrillary tangles and extracellular A␤ plaques. The plaques are composed of 42 (A␤ 1-42 )-and to a lesser extent 40 (A␤ 1-40 )-amino acid fragments of the amyloid precursor protein (1). Overproduction of the more aggregatory A␤ 1-42 peptide is believed to cause neuronal dysfunction and death in most sporadic and familial forms of Alzheimer disease. Although insoluble plaques of A␤ 1-42 are a classic feature of the brains of patients with Alzheimer disease, these plaques are also present in some healthy, elderly individuals (2). This discrepancy, and the observation that soluble A␤ is a better marker of cognitive decline (3), has led to the proposal that mature amyloid plaques are an end stage aggregation product and that the directly neurotoxic species occur earlier in the aggregation pathway (4, 5). The description of soluble oligomers of A␤ that can cause neuronal dysfunction and death (4 -6) has emphasized the importance of understanding the pathways and kinetics of A␤ aggregation. The presence of ancillary proteins that interact with A␤ may stabilize particular aggregation intermediates or seed-specific patterns of aggregation that have distinct toxic properties. Of particular interest in this regard are several proteins that are associated with ␤ amyloid plaques such as apolipoprotein E (7) and the serine protease inhibitor (serpin) ␣ 1 -antichymotrypsin (8). The role of these proteins is underscored by the finding that the E4 polymorphism of apolipoprotein E is the most powerful genetic risk factor for the development of sporadic Alzheimer disease (9, 10), most likely because it accelerates the deposition of A␤ in the brain (11-13). ␣ 1 -Antichymotrypsin is also found in the majority of senile plaques (8), with cerebrospinal fluid concentrations being consistently raised in patients with Alzheimer disease (14). Depending on the relative molar ratio, ␣ 1 -antichymotrypsin may accelerate or inhibit the aggregation of the A␤ peptide in vitro (15,16).We h...

The serpinopathies result from conformational transitions in members of the serine proteinase inhibitor superfamily with aberrant tissue deposition or loss of function. They are typified by mutants of neuroserpin that are retained within the endoplasmic reticulum of neurons as ordered polymers in association with dementia. We show here that the S49P mutant of neuroserpin that causes the dementia familial encephalopathy with neuroserpin inclusion bodies (FENIB) forms a latent species in vitro and in vivo in addition to the formation of polymers. Latent neuroserpin is thermostable and inactive as a proteinase inhibitor, but activity can be restored by refolding. Strikingly, latent S49P neuroserpin is unlike any other latent serine proteinase inhibitor (serpin) in that it spontaneously forms polymers under physiological conditions. These data provide an alternative method for the inactivation of mutant neuroserpin as a proteinase inhibitor in FENIB and demonstrate a second pathway for the formation of intracellular polymers in association with disease.Neuroserpin is a member of the serine proteinase inhibitor (serpin) 1 superfamily that is predominantly expressed by neurons in the developing and adult brain. It is secreted from the axonal growth cones of the central and peripheral nervous system, where it inhibits the enzyme tissue plasminogen activator (tPA) (1-5). The expression pattern of neuroserpin and its in vitro inhibitory activity against tPA suggest that neuroserpin has a role in controlling axonal growth, regulating emotional behavior and memory, reducing epileptic seizure activity, and limiting damage in cerebral infarction (6 -10). We have recently described an autosomal dominant dementia, FENIB, that is characterized by inclusions of mutant neuroserpin as Collins' bodies within cortical and subcortical neurons (11). This dementia is unusual in that the inclusions result from the retention of ordered polymers of neuroserpin within the endoplasmic reticulum of neurons (4,12,13). Moreover, the number of inclusions is directly related to the rate of polymer formation and inversely proportional to the age of onset of dementia (4,(12)(13)(14). For example, the Syracuse mutation (S49P) causes dementia in middle age, whereas the more rapidly polymerizing Portland mutant (S52R) causes more inclusions and an onset of dementia in the early twenties.Polymers of the serpins result from the sequential linkage between the reactive center loop of one molecule and the ␤-sheet A of another (4,(11)(12)(13)(15)(16)(17). The polymers that form in FENIB are identical to those that are formed by a mutant of another member of the serpin superfamily, the Z variant of ␣ 1 -antitrypsin (E342K), in the endoplasmic reticulum of hepatocytes in association with liver disease (15). This common mechanism of disease has allowed us to group these conditions and others that result from polymerization of serpin mutants, angio-edema (C1-inhibitor), thrombosis (antithrombin), and emphysema (␣ 1 -antichymotrypsin), as the serpinopathies (18...