One potential factor contributing to the susceptibility of these cells to premature death arises from the cytotoxic effects of amyloid-␤ (A␤) peptide deposition at or near sites of neuronal degeneration. Cultured human Franc ¸ois G.

Fatal familial insomnia (FFI) and a subtype of familial Creutzfeldt-Jakob disease (CJD), two clinically and pathologically distinct diseases, are linked to the same mutation at codon 178 (Asn178) of the prion protein gene. The possibility that a second genetic component modified the phenotypic expression of the Asn178 mutation was investigated. FFI and the familial CJD subtype segregated with different genotypes determined by the Asn178 mutation and the methionine-valine polymorphism at codon 129. The Met129, Asn178 allele segregated with FFI in all 15 affected members of five kindreds whereas the Val129, Asn178 allele segregated with the familial CJD subtype in all 15 affected members of six kindreds. Thus, two distinct disease phenotypes linked to a single pathogenic mutation can be determined by a common polymorphism.

Previously, we have shown that caspase-6 but not caspase-3 is activated by serum deprivation and induces a protracted cell death in primary cultures of human neurons (LeBlanc AC, Liu H, Goodyer C, Bergeron C, Hammond J: Caspase-6 role in apoptosis of human neurons, amyloidogenesis and Alzheimer's disease. J Biol Chem 1999, 274:23426-23436 and Zhang Y, Goodyer C, LeBlanc A: Selective and protracted apoptosis in human primary neurons microinjected with active caspase-3, -6, -7, and -8. J Neurosci 2000, 20:8384-8389). Here, we show with neoepitope antibodies that the p20 subunit of active caspase-6 increases twofold to threefold in the affected temporal and frontal cortex but not in the unaffected cerebellum of Alzheimer's disease brains and is present in neurofibrillary tangles, neuropil threads, and the neuritic plaques. Furthermore, a neoepitope antibody to caspase-6-cleaved Tau strongly detects intracellular tangles, extracellular tangles, pretangles, neuropil threads, and neuritic plaques. Immunoreactivity with both antibodies in pretangles indicates that the caspase-6 is active early in the pathogenesis of Alzheimer's disease. In contrast to the nuclear and cytosolic localization of active caspase-6 in apoptotic neurons of fetal and adult ischemic brains, the active caspase-6 in Alzheimer's disease brains is sequestered into the tangles or neurites. The localization of active caspase-6 may strongly jeopardize the structural integrity of the neuronal cytoskeletal system leading to inescapable neuronal dysfunction and eventual cell death in Alzheimer's disease neurons. Our results suggest that active caspase-6 is strongly implicated in human neuronal degeneration and apoptosis.

Extracellular amyloid β peptides (Aβs) have long been thought to be a primary cause of Alzheimer's disease (AD). Now, detection of intracellular neuronal Aβ1–42 accumulation before extracellular Aβ deposits questions the relevance of intracellular peptides in AD. In the present study, we directly address whether intracellular Aβ is toxic to human neurons. Microinjections of Aβ1–42 peptide or a cDNA-expressing cytosolic Aβ1–42 rapidly induces cell death of primary human neurons. In contrast, Aβ1–40, Aβ40–1, or Aβ42–1 peptides, and cDNAs expressing cytosolic Aβ1–40 or secreted Aβ1–42 and Aβ1–40, are not toxic. As little as a 1-pM concentration or 1500 molecules/cell of Aβ1–42 peptides is neurotoxic. The nonfibrillized and fibrillized Aβ1–42 peptides are equally toxic. In contrast, Aβ1–42 peptides are not toxic to human primary astrocytes, neuronal, and nonneuronal cell lines. Inhibition of de novo protein synthesis protects against Aβ1–42 toxicity, indicating that programmed cell death is involved. Bcl-2, Bax-neutralizing antibodies, cDNA expression of a p53R273H dominant negative mutant, and caspase inhibitors prevent Aβ1–42-mediated human neuronal cell death. Taken together, our data directly demonstrate that intracellular Aβ1–42 is selectively cytotoxic to human neurons through the p53–Bax cell death pathway.

The function of the cellular prion protein (PrP) is still poorly understood. We present here an unprecedented role for PrP against Bax-mediated neuronal apoptosis and show that PrP potently inhibits Bax-induced cell death in human primary neurons. Deletion of four octapeptide repeats of PrP (PrP⌬OR) and familial D178N and T183A PrP mutations completely or partially eliminate the neuroprotective effect of PrP. PrP remains anti-apoptotic despite truncation of the glycosylphosphatidylinositol (GPI) anchor signal peptide, indicating that the neuroprotective form of PrP does not require the abundant cell surface GPI-anchored PrP. Our results implicate PrP as a potent and novel anti-apoptotic protein against Bax-mediated cell death.Prion protein (PrP) 1 is a sialoglycoprotein that is highly expressed in brain, heart, lungs, and lymphoid system and at lower levels in several other tissues such as muscle (1, 2). Mature PrP contains two N-linked glycans and a disulfide bond (reviewed in Ref.3). PrP possesses a C-terminal GPI-anchoring signal and a transmembrane domain that can generate type I ( Ctm PrP) or type II ( Ntm PrP) transmembrane-spanning isoforms in isolated endoplasmic reticulum microsomes or phospholiposomes (4 -7). In most cells, the majority of the PrP localizes to the cell surface as a GPI-anchored protein (8). The complete translocation of PrP is dependent on translocation accessory factors (TrAF). In the absence of TrAF, PrP is exclusively synthesized in a transmembrane topology (7).Whereas the role of the infectious form of PrP in a number of human and animal neurodegenerative diseases has been extensively studied, the normal function of PrP is still poorly understood. PrP-null mice display no dramatic phenotype (9). However, evidence indicates that PrP may promote sleep continuity (10). PrP is involved in the regulation of presynaptic copper concentration, intracellular calcium concentration, activation of lymphocytes, astrocyte proliferation, and signal transduction and has antioxidant properties (11-16). Although controversial, PrP-null mice are also found to be impaired in long term potentiation (17)(18)(19). In addition, it has been shown that PrP-null neuronal cell lines are more susceptible to serum deprivation-induced cell death and that Bcl-2 overexpression can attenuate the sensitivity of PrP-null neuronal cell lines to serum deprivation (20). Kurschner and Morgan (21, 22) reported that yeast PrP fusion proteins interact with Bcl-2. Furthermore, four identical N-terminal PrP octapeptide repeats (OR) that are highly conserved in evolution share limited similarity with the Bcl-2 homology domain 2 (BH2) of Bcl-2 proteins (23, 24). Bcl-2 proteins are central to the regulation of cell death, and the BH2 domain is crucial to the anti-apoptotic function of Bcl-2 and its interaction with the pro-apoptotic Bax protein (24,25). Based on these features of PrP, we hypothesized that similar to Bcl-2 family members, PrP may play a role in the regulation of neuronal apoptosis. In the present study, we have...

Neuronal cell death, neurofibrillary tangles, and amyloid ␤ peptide (A␤) deposition depict Alzheimer's disease (AD) pathology, but neuronal loss correlates best with dementia. We have shown that increased production of A␤ is a consequence of neuronal apoptosis, suggesting that apoptosis activates proteases involved in amyloid precursor protein (APP) processing. Here, we investigate key effectors of cell death, caspases, in human neuronal apoptosis and APP processing. We find that caspase-6 is activated and responsible for neuronal apoptosis by serum deprivation. Caspase-6 activity precedes the time of commitment to neuronal apoptosis by 10 h, indicating possible activity without subsequent apoptosis. Inhibition of caspase-6 activity prevents serum deprivation-mediated increase of A␤. Caspase-6 directly cleaves APP at the C terminus and generates a C-terminal fragment of 3 kDa (Capp3) and an A␤-containing 6.5-kDa fragment, Capp6.5, that increases in serum-deprived neurons. A pulse-chase experiment reveals a precursor-product relationship between Capp6.5, intracellular A␤, and secreted A␤, indicating a potential alternate amyloidogenic pathway. Caspase-6 proenzyme is present in adult human brain tissue, and the p10 active caspase-6 fragment is detected in AD brain tissue. These results indicate a possible alternate pathway for APP amyloidogenic processing in human neurons and a potential implication for this pathway in the neuronal demise of AD. Neuronal loss distinguishes Alzheimer's disease (AD)1 from normal aging and correlates best with cognitive decline in AD individuals (Ref. 1, and reviewed in Ref. 2). In mild cases of AD, there is already a 50% loss of neurons in the entorhinal cortex, which forms the connections necessary for memory and learning between the hippocampus and the neocortex (3). Neuronal loss in AD is accompanied by the deposition of amyloid ␤ peptide (A␤) in senile plaques and cerebrovascular tissue and the presence of neurofibrillary tangles. Generally, A␤ deposition is considered important in AD. Increased production of an A␤ of 40 (A␤1-40) or 42 (A␤1-42) amino acids in length, which arises through proteolytic processing of the amyloid precursor protein (APP), is common to all familial forms of AD whether caused by mutations of APP, presenilin I, or presenilin II genes (reviewed in Ref. 4). The etiology of the most common sporadic form of the disease, which includes approximately 90% of AD cases, remains unknown. Whereas the pathology of sporadic cases of AD is identical to that of familial AD, the reason for increased A␤ in sporadic AD is unclear. We have previously shown that human primary neuron cultures committed to apoptosis produce 2-4-fold more A␤ than healthy neurons (5). Therefore, in sporadic AD, initiation of a neuronal cell death program may contribute significantly to the increased production of A␤. It is also possible that neuronal apoptosis contributes to increased A␤ in familial AD cases because overexpression of APP, and mutations of APP or presenilin, induce neuronal cell...

Fatal familial insomnia is a prion disease with a mutation in codon 178 of the PrP gene, but the disease phenotype seems to differ from that of previously described kindreds with the same point mutation.

Estrogen is an active neuroprotectant and is presently investigated as a potential therapy against Alzheimer's disease for women. To determine if male hormones could also be neuroprotective, we investigated the effect of testosterone, methyltestosterone, and epitestosterone at physiological concentrations on primary cultures of human neurons induced to undergo apoptosis by serum deprivation. Serum deprivation signi®cantly induces neuronal apoptosis in a protracted fashion. As expected, physiological concentrations of 17-bestradiol and transcriptionally inactive 17-a-estradiol protect neurons against apoptosis. Similar to 17-b-estradiol, physiological concentrations of testosterone are also neuroprotective. Androgen receptors are present at 8^2 fmol/mg protein in the neuron cultures. The non-aromatizable androgen, mibolerone, is also neuroprotective and aromatase inhibitor, 4-androsten-4-OL-3,17-dione, does not prevent testosteronemediated neuroprotection. In contrast, anti-androgen,¯utamide, eliminates testosterone-mediated neuroprotection. Testosterone analog, methyltestosterone, showed androgen receptordependent neuroprotection that was delayed in time indicating that a metabolite may be the active agent. The endogenous anti-androgen, epitestosterone, also showed a slight neuroprotective effect but not through the androgen receptor. These results indicate that androgens induce neuroprotection directly through the androgen receptor. These data suggest that androgens may also be of therapeutic value against Alzheimer's disease in aging males.