C99 selectively accumulates in vulnerable neurons in Alzheimer's disease

The levels and distribution of amyloid deposits in the brain does not correlate well with Alzheimer's disease (AD) progression. Therefore, it is likely that amyloid precursor protein and its proteolytic fragments other than amyloid b (Ab) contribute to the onset of AD.


INTRODUCTION
Alzheimer's disease (AD) is the most common type of dementia, characterized by accumulation of extracellular and intracellular amyloid b (Ab) aggregates, intracellular paired helical fragments and extensive age-dependent neuronal loss in vulnerable areas. [1][2][3] Robust genetic and biochemical evidence points to alterations of amyloid precursor protein (APP) metabolism as causative in the development of AD. In the amyloidogenic pathway, APP is cleaved by -secretase (BACE) to generate soluble APP beta protein (sAPPb) and C99. -secretase then cleaves C99 to generate A and amyloid precursor protein intracellular domain (AICD). Accumulation of A is a cardinal feature of AD, but its role in causing injury in AD remains unclear. Thus, the distribution of probes added to the reaction bind to the amplified DNA. The resulting fluorescent signal can be detected by fluorescence microscopy. This technique offers great sensitivity because powerful rolling circle amplification is used. It also offers great selectivity because dual target recognition is used. 10,11 Here, we used antibodies generated against both the N and C terminal domains of C99 in cultured cells and found that we can distinguish C99 from Ab, full-length APP, C83, and AICD. The adaptation of the proximity ligation assay for the detection of C99 provides a general framework for the detection in situ of low-abundance proteolytic fragments in the human brain and animal models.
Secondary antibody was AlexaFluor 488 from Molecular Probes.  fit for K0, and amplitude values were evaluated to ensure the quality of the reconstruction. The reconstruction was then subjected to further processing by the "OMX Align Image" function. The version of the final image was then compared with a widefield version of the same image using the "Generate Widefield" and "Deconvolve" functions on the raw nonreconstructed data. Individual sections from the total z-stack were used for comparison.

Proximity ligation assay in human brain sections
Forty-nine human brain slides from the hippocampus, frontal cortex, and occipital cortex were obtained from the brain bank at Mount Sinai/JJ Peters VA Medical Center NIH Brain and Tissue Repository.
Donors were selected to evidence no neuropathology or only the neuropathology of AD excluding any other discernable neuropathology, such as cerebrovascular disease or Lewy bodies. Before proceeding with the staining protocol, the slides were deparaffinized and rehydrated. Slides were placed in a rack, and the following washes were per-

PLA quantification
C99-PLA produces bright red dots that were quantified using the Image J software (NIH). The tiff images were thresholded, and the function "Analyze particles" was applied in the red channel. In the blue channel, corresponding to DAPI staining, the function "Cell counter" was applied after threshold.

Amyloid plaque staining
Sections from paraffin-embedded blocks were stained using hematoxylin-eosin and anti-amyloid 4G8 and anti-tau AD2. All neuropathologic data regarding the extent and distribution of neuropathologic lesions were collected by neuropathologists unaware of any of the clinical and psychometric data.
For quantitative measures of plaque density, 5 representative highpower fields (0.5 mm 2 ) were examined and an average density score was calculated for each region and expressed as mean plaque density per square millimeter. When plaques were unevenly distributed in each slide, plaques in the region with the highest density were counted.
These sections were adjacent to the sections used to quantify C99 levels.

Statistical analysis
The data are presented as means ± SD. The significance level was determined using a two-way ANOVA with the Bonferroni post-test,

RESULTS
First, we screened several antibodies aimed at recognizing the N-and C-terminal domains of C99. The combination of 6C3, a mouse monoclonal antibody 12 which recognizes the cleaved N-terminal of C99 and RU-369, a rabbit polyclonal antibody 13 designed to recognize the Cterminal of APP, successfully recognized C99 (Fig. 1A). As expected with a C99-specific marker, the signal was greatly reduced by a BACE inhibitor, which blocks C99 synthesis, and dramatically increased by a -secretase inhibitor, which blocks C99 breakdown (Fig. 1B) Fig. 1). C99-PLA produces brightred dots that can easily be quantified. Super-resolution microscopy revealed that each dot, measuring about 200400 nm, consisted of a group of 2-3 smaller dots, consistent with the idea of aggregation of C99 molecules (Fig. 1C).
Next, we applied this PLA methodology to study the localization of C99 in the human-brain tissue. Brain tissue sections were obtained from the hippocampus, frontal cortex, and occipital cortex from 12 controls and 37 patients suffering from sporadic AD. Age at the time of death, sex, Braak stage, Thai, and the CERAD score of these subjects is listed in Supplementary Table 1. PLA specificity was verified by omitting one of the antibodies (Supplementary Fig. 2). As seen in Fig. 2A (Fig. 2B, E). In the middle frontal gyrus of the frontal cortex, C99 levels were increased when comparing patients with severe AD with controls (7.1 ± 2.0 vs. 2.3 ± 1.6, P < .001) and patients with severe AD with patients with moderate AD (7.1 6 2.0 vs. 2.60 6 1.6, P < .001) (Fig. 2C, E). Levels of C99 in the occipital cortex, an area considered to be resistant to neurodegeneration, were similar in all three groups (Fig. 2D, E). Four additional samples from the hippocampus of nondemented patients and patients with severe AD were subjected to Western blot analysis using the antibody 6E10. An increase of 4.5 times in C99 levels was observed when comparing patients with severe AD with nondemented controls, comparable to the increase seen by PLA ( Supplementary   Fig. 3) with AD in the dentate gyrus and frontal cortex ( Fig. 3A and C). Interestingly, C99 also accumulated in astrocytes in patients with AD, where it was localized in both the soma and processes, as revealed by glial fibrillary acidic protein (GFAP) staining. However, in contrast to neurons, the distribution of C99 in astrocytes did not change between nondemented and demented patients ( Fig. 3B and C). Next, we compared the levels of A plaques and C99 in the middle frontal cortex and  Fig. 4A and B, A plaques were present in both vulnerable and nonvulnerable areas, whereas C99 accumulation was correlated with disease progression only in the middle frontal cortex, a vulnerable area, but not in the occipital cortex, a resistant area.
To study the association of C99 with selective neuronal vulnerability, co-staining with tau was performed in the CA1 area of 7 nondemented controls and 15 patients with AD. As seen in Fig. 4C and D, C99 levels were greatly increased in neurons with high levels of tau, which is a widely accepted marker of neuronal vulnerability. These results show that C99 accumulation, as opposed to A plaques, is specific to neurons most vulnerable to neurodegeneration in AD.

DISCUSSION
AD is characterized by accumulation of amyloid plaques and paired helical filaments. Mutations that cause early-onset AD are located in the genes that code for the APP and the enzyme responsible for amyloid production, Presenilin 1/Presenilin 2. These observations have led to the widely-held belief that A has a causative role in the development of AD. However, the distribution of amyloid deposits in the brain does not correlate well with disease progression. These results suggest the possibility that APP metabolites other than A might contribute to AD. It has been proposed that C99, a specific APP metabolite, may cause neurodegeneration by inducing endosomal enlargement, 14,15 leading to endosomal dysfunction and neuronal death. [16][17][18][19] In this study, we have developed a method to detect C99 in situ and applied this method to measure the levels of C99 in postmortem human-brain samples of individuals at different stages of AD dementia. We found a correlation between levels of C99 and levels of cogni-  14,15 It will be interesting to see the specific sites of C99 accumulation in the human brain (for instance, using PLA and various vesicle markers) and the effect of such accumulation on the endosomal-lysosomal and autophagic pathways in human neurons in culture. It has also been shown that C99 accumulation in the mouse brain leads to an increase in vesicles positive for Cathepsin B and Lamp1, as well as increased levels of LC3II and autophagic dysfunction. 17 Although the exact molecular mechanism by which C99 exerts this effect is not yet known, it is becoming clear that C99 accumulation contributes to neuronal dysfunction and ultimately to neuronal death.
Our results show a correlation between C99 levels and cognitive impairment. It has been previously found that synaptic loss is the major correlate of cognitive impairment. 4 To analyze the relationship of C99 with synaptic loss, it could be possible to measure synaptic markers like SNAP25 and synaptophysin, 35 in both vulnerable and resistant brain areas and correlate them with C99 levels.
The method that we developed to detect specific APP fragments in fixed brain slides can be modified to quantify APP metabolites in human fluids. To that end, it could be possible to quantify C99 levels in plasma and CSF and correlate them with cognitive deterioration and vulnerability to neurodegeneration, with the goal of developing C99 as an AD biomarker.
In conclusion, our results raise the possibility that C99, rather than A , is responsible for the death of nerve cells in AD. In that case, bsecretase inhibitors, which lower both C99 and A , are preferable to -secretase inhibitors, which lower A but increase C99 levels.

ACKNOWLEDGMENTS
The authors thank Dr.