a.vi.i. Late onset AD genes involved in inflammation

No documento Peter St George-Hyslop (páginas 52-56)

As noted in the previous paragraph, genetic discoveries about the in-nate immune and inflammatory pathway genes (described in detail below) are extremely important discoveries because these pathways potentially rich intractable diagnostic and therapeutic targets that might be exploited for pa-tient benefit.

Preliminary studies reveal that CD33 is expressed in microglial cells. In AD cases the CD33-positive microglia are activated (Figure 16). CD33 is a member of a major subfamily of sialic acid binding immunoglobulin like lec-tins168. CD33, like other members of this family have immune receptor tyro-sine-based inhibitory motifs and signal negatively to Toll-like receptors during innate immune responses. CD33 acts as an endocytic receptor,

mediat-TRANSLATING DISCOVERIES IN BASIC MOLECULAR BIOLOGY, CELL BIOLOGY, AND MOLECULAR GENETICS INTO TRANSFORMATIVE APPROACHES TO THE DIAGNOSIS AND TREATMENT OF CURRENTLY INCURABLE NEURODEGENERATIVE DEMENTIAS

ing endocytosis through a mechanism independent of clathrin. CD33 is known to be involved in apoptosis during an immune response169. Mutational analysis of this gene is ongoing, but our working hypothesis is that sequence variants in CD33 alter microglial and bone marrow derived macrophage function in-volved in the uptake and clearance of extracellular Aβ and tau aggregates.

CR1 (complement receptor 1) is a glycoprotein expressed on erythro-cytes, leucocytes and follicular dendritic cells, and mediates cellular bind-ing to particles and immune complexes that have activated the complement pathway (especially complement factors C3b and C4b-opsonised foreign antigens). In this role, it mediates immune adherence and phagocytosis. CR1 can also act as a negative regulator of the classic and alternative

comple-Figure 16: CD33

CD33 Control

CD33 stains microglial cells (arrows) in both the control and AD cases. In AD cases the CD33-positive microglia are activated as shown by increased cytoplasm.

AD

Figure 17: CR1

CR1 (CD35) Control

CR1(CD35) stains strongly the choroid plexus in the control and AD cases (not shown). In AD cases, astrocytes (middle) and microglia (right) are also stained.

AD AD

DISCOVERIES ARISING FROM BASIC SCIENCE STUDIES OF SPECIFIC NEURODEGENERATIVE DEMENTIAS

ment pathways. CR1 is expressed in the choroid plexus, but not in the brain parenchyma (Figure 17). In AD cases, astrocytes and microglia are strongly stained with CR1 antibodies. Intriguingly, CR1 genotype is associated with the endophenotype of amyloid plaque burden170. Our mutational analyses have identified that the pathogenic allele in CR1 is the duplication of a low complexity repeat motif (CR1-S)171. This coding sequence insert alters the intracellular processing of the longer isoform, causing it to be retained in the ER, and resulting in less CR1 at the cell surface (unpublished). Consequently, the AD-associated allele in this innate immune/inflammatory pathway gene likely also has a deleterious effect on innate immune/inflammation-mediated removal of extracellular aggregates of Aβ and tau etc.

We have shown that CD2AP stains vessel walls in control brains, whereas in AD there is also strong microglial labelling (Figure 18). CD2AP is an en-docytosis associated protein which interacts with the clathrin scaffold during clathrin-mediated endocytosis. Mutants in CD2AP are associated with ne-phrotic syndrome. However, CD2AP has also been implicated in dynamic ac-tin remodeling and membrane trafficking/receptor patterning in T-cells and is capable of modulating T-cell receptor signaling172 173. CD2AP is also involved in cytotoxic processes by Natural Killer (NK) cells172. Mutational analyses are underway. Functional studies suggest that knockdown of CD2AP is associated with impaired microglial activation in the presence of Aβ (manuscript in prep-aration). These studies again suggest that loss of function alleles in CD2AP result in an impairment of innate immune/inflammation-mediated clearance of neurotoxic extracellular protein aggregates.

Figure 18: CD2AP

CD2AP Control

CD2AP stains vessel walls in control brains, whereas in AD there is also extensive and strong microglial labeling . The vascular staining is maintained in AD (not shown)

AD

TRANSLATING DISCOVERIES IN BASIC MOLECULAR BIOLOGY, CELL BIOLOGY, AND MOLECULAR GENETICS INTO TRANSFORMATIVE APPROACHES TO THE DIAGNOSIS AND TREATMENT OF CURRENTLY INCURABLE NEURODEGENERATIVE DEMENTIAS

We detected that CLU (clusterin) labels small punctate cytoplasmic “in-clusions” in neurons. These “in“in-clusions” become more profuse and more numerous in AD cases compare to controls (Figure 19a). In AD cases, CLU also stains plaques and neurofibrillary tangles. Mutational analysis has re-vealed two types of AD-associated variants. The most common variant is the presence of SNPs in a non-coding intronic regulatory element that regulates protein abundance (presumably by reduced mRNA transcription) (Figure 19b). Work by our colleague David Klenerman, suggests that clusterin is important for chaperoning Aβ and precluding oligomer formation174. Lower levels of clusterin might therefore be expected to encourage aggregate for-mation. The second type of AD-associated variant that we have discovered

Figure 19a: CLU

Clusterin

Control AD

Clusterin labels small punctate cytoplasmic “inclusions” in normal neurons in controls. These become more profuse and more numerous in AD cases (black arrows).

Clusterin does also stain neurfibrillary tangles (red arrow) and plaques (not shown) in AD cases.

Figure 19b: CLU

DISCOVERIES ARISING FROM BASIC SCIENCE STUDIES OF SPECIFIC NEURODEGENERATIVE DEMENTIAS

are a number of missense mutations and one in-frame nine nucleotide (three codon) deletion in the β-chain of the CLU protein175. We are currently inves-tigating the effect of these mutations. However the expectation is that they will also cause a loss of function effect on the chaperone activity of CLU, and like the other AD associated variants will reduce the removal of neuro-toxic extracellular protein aggregates.

Finally, during whole exome sequencing of affected pairs of AD cases from families with multiple AD cases (“multiplex”), we identified several families where the affected members had heterozygous missense mutations in the Triggering Receptor Expression On Myeloid cells 2 gene (TREM2)78. TREM2 is expressed on a number of cells including bone marrow-derived macrophages (microglia) and neurons. We are exploring the effect of these mutations (e.g. R47H), which are probably loss of function alleles. Our working hypothesis is that once again these variants reduce capacity to re-move extracellular neurotoxic protein aggregates of Aβ and/or tau.

III.1.a.vi.ii. Transcriptional Micro-Array Profiling of Aβ-Responsive

No documento Peter St George-Hyslop (páginas 52-56)