± 0.3 8.2\*\*\*,#,## ± 0.5

\*\* ± 3.7 58.7\*,

BM cells at 4 (young) and 22 (old)

\*\*\* ± 5.4

\*\* ± 2.1 35.2\* ± 1.3 28.3\*\*,# ± 3.2 37.3## ± 2.6

\*\* ± 0.5 15.1 ± 1.0 7.7\*\*,# ± 0.5 15.3\*\*,## ± 0.4

and to elucidate the ability of stem cells to restore the immune system of old animals.

**Parameters Experimental groups**

splenocytes, % 56.3 ± 2.3 49.4\* ± 1.9 42.3\*,

80 Molecular Mechanisms of the Aging Process and Rejuvenation

splenocytes, % 17.3 ± 1.4 13.3 ± 0.6 11.2\*,

Thy 1.2+

CD4+

CD8+

CD4+ CD44+

IgM+

groups, M ± SE.

at least 8.

Thymus mass, mg 34.6 ± 2.1 18.1\* ± 2.4 17.7\* ± 3.8 12.5\* ± 1.3 6.4\*\*,

Mice were divided into six groups: (1) young intact animals (Y), (2) young irradiated animals that were administered BM cells from young mice (Yy), (3) young irradiated animals that were administered BM cells from old mice (Yo), (4) old intact animals (O), (5) irradiated old animals that were administered BM cells from young mice (Oy), and (6) old irradiated animals that were administered BM cells from old mice (Oo). Functional status of the immune system was evaluated by the following parameters: the weight of the thymus, the number of leukocytes in the blood, the number of lymphocyte subpopulations in the spleen using flow cytometry and antibodies to the mouse antigens CD4, CD8, IgM, CD44 (Sigma, USA), and the proliferative activity of splenocytes in vitro in response to mitogens phytohemagglutinin (PHA), concana‐ valin A (Con A), and lipopolysaccharide (LPS).

It is known that the immune system of old animals is different from the young in many investigated indices. Particularly, thymus weight and the number of white blood cells are significantly reduced in old individuals. Moreover, the number of CD4+ cells and the CD4+ / CD8+ ratio in the spleen are decreased in aged animals. Lymphocytes with a phenotype of memory T-cells (CD4 + CD44+ ) increases in old animals are approximately twice higher. These changes in subpopulations of T-cells significantly affect their functional properties. For instance, lymphocytes of old animals respond poorly to T-lymphocyte antigens PHA response and Con A response. Response to LPS response (B-lymphocyte-specific mitogen) also signifi‐ cantly reduced with age, despite the high content of B-cells in the spleens of old mice CBA/Ca. Other authors [1, 5] also observed similar changes in the composition and functions of the spleen cells of old mice. We performed the experiments of heterochronic transplantation of BM cells with irradiated CBA/Ca mice. The results are shown in **Table 1** and **Figure 1**. We can find out the following interesting changes.

First, the changes were caused by transplantation procedure itself. Significant reduction in thymus weight to all exposed mice was observed possibly as a result of the effect of prolonged stress and radiation damage (**Table 1**). In young recipients, the number of T-cells in the spleen was also decreased (**Table 1**). Otherwise, in old recipients, transplantation of BM cells (irrespective of donor age) led to an increase in the proliferative response to mitogens. For example, the splenocyte response to Con A and LPS in old recipients reached the level of young mouse (**Figure 1**).

The second type of changes has occurred in an organism the young recipients after injection the old BM cells (Yo group). In particular, we observed a decrease in the content of Thy 1.2+ lymphocytes perhaps by increasing the number of CD8+ cells in the spleen (**Table 1**). Yo group showed an increased content of IgM+ cells and increased number of CD4+CD44+ memory-like cells in the spleen (**Table 1**). These data are confirmed by many other studies, which reveal HSCs dysfunctions with age [7, 8].

CBA/Ca female mice were irradiated and recovered of 15 × 106 BM cells at 4 (young) and 22 (old) months of age. After 3 months, the recovering mice were euthanized and subjected to immune parameter analysis. Symbol of the experimental groups: Y, young intact mice; Yy, young irradiated animals that were recovered of BM cells from young mice; Yo, young irradiated animals that were recovered of BM cells from old mice; O, old intact mice; Oy, old irradiated animals that were recovered of BM cells from young mice; and Oo, old irradiated animals that were recovered of BM cells from old mice.

\*, *P* (*t*) < 0.05 comparing young intact animals.

\*\*, *P* (*t*) < 0.05 comparing young irradiated animals recovered of young BM cells.

\*\*\*, *P* (*t*) < 0.05 comparing young irradiated animals recovered of old BM cells.

#, *P* (*t*) < 0.05 comparing old intact animals.

##, *P* (*t*) < 0.05 comparing old irradiated animals recovered of young BM cells.

###, *P* (*t*) < 0.05 comparing old irradiated animals recovered of old BM cells.

It should be noted that we did not detect the rejuvenation effect of a young bone marrow cells on the old immune system functions. The third change is caused by the systemic factors of organism. Major parameters—thymus mass, number of T-cells in the spleen, contents of CD4+ lymphocytes, response to T-cell mitogens, number of IgM+, and CD4+ CD44+ lymphocytes —in young recipients remained as high as those in young intact individual. Mice of Oy group showed lower weight of the thymus and decreased number of CD8+ cells in the spleen compared to animal group Oo (**Table 1** and **Figure 1**). The obtained data may suggest that young hematopoietic cells may decrease the ability for their comprehensive development in old recipients compared to their own old hematopoietic cells (**Table 1** and **Figure 1**). Further‐ more, an increased level of CD4+ CD44+ splenocytes in Oy mice is an interesting fact. It is known that the number of T-cells with memory phenotype steadily increases with age. It is supposed that the differentiation of naïve T-lymphocytes in memory T-cells can be induced by repeating antigenic load as well as a chronic inflammation (inflammaging) in the old body [9]. Therefore, high level of memory-like cells in old recipient may suggest that the macroenvironment of old organism can specifically stimulate the differentiation and accumulation of cells with memory phenotype, regardless of the "age" of hematopoietic stem cells.

The results of this study not only confirmed the known information about the changes of the immune system with aging but also gave new information about the possible mechanisms of age-related dysfunction. It should be noted that the residence of young cells/tissues in an old body leads to a deterioration of their functional properties in most studies using heterochronic chimeras [12–15].

In our study of young hematopoietic cells were also unable to improve any of the investigated parameters of the immune system in old animals. Thus, we examined the number of antibodyforming cells to sheep red blood cells (SRBCs). The immune response to thymus-dependent antigens is not affected by the age of HSCs, but depended on the age of the recipients (**Figure 2**). In young recipients, on the contrary, the immune response of Yy and Yo showed similar strength to that of young intact mice (Y). This fact may suggest that the age-related change of HSCs functions is not a key factor in the occurrence of age-related dysfunction of the immune system.

young irradiated animals that were recovered of BM cells from young mice; Yo, young irradiated animals that were recovered of BM cells from old mice; O, old intact mice; Oy, old irradiated animals that were recovered of BM cells from young mice; and Oo, old irradiated

It should be noted that we did not detect the rejuvenation effect of a young bone marrow cells on the old immune system functions. The third change is caused by the systemic factors of organism. Major parameters—thymus mass, number of T-cells in the spleen, contents of

—in young recipients remained as high as those in young intact individual. Mice of Oy group showed lower weight of the thymus and decreased number of CD8+ cells in the spleen compared to animal group Oo (**Table 1** and **Figure 1**). The obtained data may suggest that young hematopoietic cells may decrease the ability for their comprehensive development in old recipients compared to their own old hematopoietic cells (**Table 1** and **Figure 1**). Further‐

that the number of T-cells with memory phenotype steadily increases with age. It is supposed that the differentiation of naïve T-lymphocytes in memory T-cells can be induced by repeating antigenic load as well as a chronic inflammation (inflammaging) in the old body [9]. Therefore, high level of memory-like cells in old recipient may suggest that the macroenvironment of old organism can specifically stimulate the differentiation and accumulation of cells with memory

The results of this study not only confirmed the known information about the changes of the immune system with aging but also gave new information about the possible mechanisms of age-related dysfunction. It should be noted that the residence of young cells/tissues in an old body leads to a deterioration of their functional properties in most studies using heterochronic

In our study of young hematopoietic cells were also unable to improve any of the investigated parameters of the immune system in old animals. Thus, we examined the number of antibodyforming cells to sheep red blood cells (SRBCs). The immune response to thymus-dependent antigens is not affected by the age of HSCs, but depended on the age of the recipients (**Figure 2**). In young recipients, on the contrary, the immune response of Yy and Yo showed similar strength to that of young intact mice (Y). This fact may suggest that the age-related change of HSCs functions is not a key factor in the occurrence of age-related dysfunction of

CD44+

splenocytes in Oy mice is an interesting fact. It is known

lymphocytes

\*\*, *P* (*t*) < 0.05 comparing young irradiated animals recovered of young BM cells.

\*\*\*, *P* (*t*) < 0.05 comparing young irradiated animals recovered of old BM cells.

##, *P* (*t*) < 0.05 comparing old irradiated animals recovered of young BM cells.

lymphocytes, response to T-cell mitogens, number of IgM+, and CD4+

CD44+

phenotype, regardless of the "age" of hematopoietic stem cells.

###, *P* (*t*) < 0.05 comparing old irradiated animals recovered of old BM cells.

animals that were recovered of BM cells from old mice.

\*, *P* (*t*) < 0.05 comparing young intact animals.

82 Molecular Mechanisms of the Aging Process and Rejuvenation

#, *P* (*t*) < 0.05 comparing old intact animals.

more, an increased level of CD4+

chimeras [12–15].

the immune system.

CD4+

**Figure 2.** The number of antibody-forming cells to SRBCs in the spleen of immunized CBA/Ca experimental mice.Sym‐ bol of the experimental groups: Y, young intact mice; Yy, young irradiated animals which were recovered of BM cells from young mice; Yo, young irradiated animals which were recovered of BM cells from old mice; O, old intact mice; Oy, old irradiated animals which were recovered of BM cells from young mice. CBA/Ca female mice were irradiated and recovered of 15 × 106 BM cells at 3 (young) and 24 (old) months of age. At 3 months after the recovering mice were immunized of 108 SRBCs intraperitonealy. Four days after animals were euthanized and subjected to immune parame‐ ter analysis. \*, *P* (*t*) < 0.05 comparing young intact animals; #, *P* (*t*) < 0.05 comparing old intact animals. The mice num‐ ber in each group is at least 8.

Consequently, we could identify the following age-related differences in the regenerative properties of HSCs. HSCs of old mice have a reduced ability to produce Thy 1.2+ cells, CD4+ , and CD8+ cells but fully recovered the ability to develop an immune response to SRBCs in young recipients. Transplantation of HSCs of old mice results in the opposite effect in old individuals: it led to accumulation of more Thy 1.2+ and CD8+ cells, and more significant proliferative response of splenocytes to PHA was observed (**Figure 1** and **Table 1**). However, these changes do not result in the restoration of immune response to SRBCs. These results may suggest that the differentiation of HSCs is affected by some factors present in the microenvir‐ onment of lymphoid organs, where HSCs are maturated. To elucidate this hypothesis, we used another model—heterochronic parabiosis—which is described in the next chapter.

#### **2.2. Study of the mechanisms of age-related changes in the immune system on the model heterochronic parabiosis**

The parabiosis model represents a pair of animals in which the common blood circulation is established by a surgical procedure. In fact, this model allows creating Siamese twins surgically in experimental conditions [16]. The model of parabiosis is actively studied in recent years. It was shown that the mutual exchange of progenitor cells of fibroblast/myofibroblasts [17, 18], hematopoietic [19, 20] and non-hematopoietic stem cells [21], and the endothelial progenitor cells [22] actively occurs between partners due to the general bloodstream.

We evaluated the status of the immune system in heterochronic parabionts 3 months after the operation [23]. We assumed that the migration of young stem cells in combination with young serum factors will contribute to restoration of the structure and function of the thymus in old animals. The rejuvenation of the thymus, in turn, will lead to restoration of function and the repertoire of T-cells in peripheral lymphoid organs and the rejuvenation of the immune system of old parabiotic partners.

Disturbances of thymic function with age are well known. It is found that the age-induced thymus atrophy is accompanied by a reduction in thymocyte number and a decrease in T-cell progenitors in the bone marrow. These disturbances can be induced by changes in the hormonal balance, for example, increased levels of sex steroids with age [24]. When using the model heterochronic parabiosis, we suggested that the young blood will improve the function of the thymus in the old animals. As a result, we have not found improved indices in thymus parameters of old partners. Furthermore, we obtained a decrease in thymus weight in young heterochronic partner without significant changes in thymocyte subpopulation number [23].

To assess changes in the T-cell functions, we evaluated the parameters that are most signifi‐ cantly changed during aging: CD4 and CD8 T-cell subpopulations in the spleen, and the proliferative activity of splenocytes in response to mitogens in vitro [25]. It was shown that the proliferative activity of splenocytes in response to PHA in vitro was not restored in old heterochronic partners, and decreased in the young ones after 3 months of coexistence. Similar changes have been identified in the CD4+ and CD8+ T-cell subpopulation numbers in the spleen. The CD4/CD8 ratio did not change the spleen of old partners and significantly decreased in the young ones. Furthermore, it has been shown that the amount of the cells with memory phenotype (CD44+ ) was increased in young partners to the level of old animals (**Table 2**).

**Table 2.** Effects of heterochronic parabiosis on the immunological parameters of CBA/Ca mice, M ± SE.

hematopoietic [19, 20] and non-hematopoietic stem cells [21], and the endothelial progenitor

We evaluated the status of the immune system in heterochronic parabionts 3 months after the operation [23]. We assumed that the migration of young stem cells in combination with young serum factors will contribute to restoration of the structure and function of the thymus in old animals. The rejuvenation of the thymus, in turn, will lead to restoration of function and the repertoire of T-cells in peripheral lymphoid organs and the rejuvenation of the immune system

Disturbances of thymic function with age are well known. It is found that the age-induced thymus atrophy is accompanied by a reduction in thymocyte number and a decrease in T-cell progenitors in the bone marrow. These disturbances can be induced by changes in the hormonal balance, for example, increased levels of sex steroids with age [24]. When using the model heterochronic parabiosis, we suggested that the young blood will improve the function of the thymus in the old animals. As a result, we have not found improved indices in thymus parameters of old partners. Furthermore, we obtained a decrease in thymus weight in young heterochronic partner without significant changes in thymocyte subpopulation number [23].

To assess changes in the T-cell functions, we evaluated the parameters that are most signifi‐ cantly changed during aging: CD4 and CD8 T-cell subpopulations in the spleen, and the proliferative activity of splenocytes in response to mitogens in vitro [25]. It was shown that the proliferative activity of splenocytes in response to PHA in vitro was not restored in old heterochronic partners, and decreased in the young ones after 3 months of coexistence. Similar

and CD8+

The CD4/CD8 ratio did not change the spleen of old partners and significantly decreased in the young ones. Furthermore, it has been shown that the amount of the cells with memory

**Young heterochronic**

**(n = 8)**

Splenocytes number, 106 153.60 ± 16.56 150.00 ± 11.21 118.80 ± 9.60 90.80 ± 12.53a

Phagocytic macrophages, % 63.00 ± 2.57 50.00 ± 3.32\*\* 48.38 ± 3.75 52.40 ± 2.17aa

) was increased in young partners to the level of old animals (**Table 2**).

**8)**

17.38 ± 1.05 14.88 ± 1.39 14.81 ± 2.26 17.81 ± 1.06

1.71 ± 0.15 1.32 ± 0.13\* 1.59 ± 0.25 1.39 ± 0.07a

22.10 ± 0.91 19.16 ± 1.93 22.54 ± 2.36 20.63 ± 2.16

9.75 ± 0.91 16.23 ± 1.75\*\* 15.34 ± 0.54 12.70 ± 1.22

T-cell subpopulation numbers in the spleen.

**Old heterochronic (n =**

**Old isochronic (n =**

**10)**

cells [22] actively occurs between partners due to the general bloodstream.

of old parabiotic partners.

84 Molecular Mechanisms of the Aging Process and Rejuvenation

changes have been identified in the CD4+

**Parameters Experimental groups**

**Young isochronic**

**(n = 8)**

phenotype (CD44+

Bone marrow cell number, 106

Proliferative activity in vitro,

cell number in the

cell number in the

per femur

CD4+

CD8+

spleen, %

spleen, %

stimulation index

For the parabiosis, CBA/Ca male mice were typically joined at 2–3 (young) and 22–23 (old) months of age. At 3 months after the parabiosis initiation, mice were euthanized and subjected to immune parameter analysis. All pairs with tumors were excluded from the study. \*, *p*(*t*) < 0.05 comparing isochronic animals of the same age; \*\*, *p*(*t*) < 0.01 comparing isochronic animals of the same age, a, *p*(*t*) < 0.05 comparing young isochronic animals; aa, *p*(*t*) < 0.01 comparing young isochronic animals.

The most optimistic result in such a pessimistic picture was shown by regulatory T-cells—their number decreased significantly in the old heterochronic partners to young level (**Table 2**).

Thus, at this stage of study it was found that the mutual exchange of blood between hetero‐ chronic partners did not result in recovery of the thymus, the peripheral T-cell number, and immune functions normalizing in aged parabiotic partners. Major changes have been observed in young partners. There was a decrease of thymus weight, disorders of peripheral T-cell repertoire, and a decrease in PHA-stimulated lymphocyte proliferation in vitro. In fact, we observed age-related changes in the immune system of young heterochronic partners in accelerated rate.

This model of accelerated aging of the immune system has provided us with a unique opportunity to trace the dynamics of the appearance of age-related changes in the immune system. We examined the changes in immune functions in young heterochronic partners at different time points—3, 6, and 12 weeks after surgery. We found that the first change, which occurs in young heterochronic partners, was doubling number of CD8+ 44+ cells in the spleen 3 weeks after surgery. After 6 weeks, the rate of this index remained at a high level, and there was also a significant decrease in CD4/CD8 ratio in the spleen. At the 12th week of coexistence, a decrease in the proliferative response of T-cells in response to PHA, as well as significantly decreased number of phagocytic cells in the spleen, was found [23].

Increasing the number of CD8+ 44+ memory-like cells in the spleen is the first change of a young partner. The study of lymphoid cell migration between the heterochronic partners has shown that an increasing number of CD8+ 44+ cells may be mediated by an increase in the number of young CD8+ 44+ cells in the spleen, bone marrow, and thymus of old partners [23]. We hy‐ pothesized that these changes may have multiple mechanisms of occurrence: the migration of young CD8+ 44+ cells to the old partner, or the proliferation of young cells into the old body through antigen-specific or homeostatic proliferation. However, we cannot assert which of the proposed mechanisms is the key in age-related changes on the basis of the obtained results.

The population of CD8+ 44+ memory-like cells is short-lived and quickly proliferating. It is assumed that a shorter time CD8+ 44+ T-cell recycling relative to naïve CD8+ T-cells is associated with the higher speed of their homeostatic proliferation [26]. At the same time, in aged mice, the reduction in the turnover of memory-like CD44+ CD8+ cells was found. The authors suggested that the reduced turnover of aged CD44+ CD8+ cells reflected an inhibitory influence of the aged environment, including APCs [27–29]. The presented data may indicate that the old lymphoid microenvironment can induce changes in the differentiation and functioning of CD8+ T-cells. We suggested that the rapid growth of the number of CD8+ 44+ cells in young heterochronic partners may result from the mutual influence of the two environments—young for fast proliferation and old for CD8+ 44+ memory cell differentiation.

The obtained data indicate that our hypothesis—that the mechanisms of the immune aging can be mediated by dysfunctions of lymphoid cells and organs (thymus, bone marrow) or insufficient production of some factors (hormones, cytokines, and growth factors)—is un‐ founded. The obtained results showed that the general circulation between partners of different ages, which provides a mutual exchange of hematopoietic stem cells, naïve lympho‐ cytes, hormones, cytokines, and growth factors, does not lead to the restoration of the old immune system functioning. On the contrary, it was found that the common bloodstream from the old to the young partner is able to induce the aging of a young immune system. It is assumed that changes in the proliferation and differentiation of young CD8+ T-cells, which presumably enhanced by their homeostatic proliferation, appear due to the influence of the old lymphoid niches. To test this hypothesis, we used the model of heterochronic cocultivation of lympho‐ cytes with cells that form spleen lymphoid niche (macrophages or CD 11c+ dendritic cells).

#### **2.3. The study of age-related changes of lymphoid niche cells in the model of heterochronic parabiosis**

It is well known that peripheral T-cell functions are regulated by multiple extrinsic factors, in particular by the microenvironment of lymphoid organs, where T-cells usually form special zones such as a periarteriolar lymphoid sheath in the spleen. T-cell activities in these zones are regulated by cells of their niche. Lymphoid niche includes fibroblastic reticular cells, which organize T-cell zones and antigen-presenting cells. APCs are important T-cell regulators and include a broad variety of different cell types such as dendritic cells, B-cells, and macrophages. Dendritic cells are professional APCs and regulatory cells, and are able to either induce tolerance or stimulate profound immune response in an organism [30]. Macrophages can also display some DC-like features [31]. APCs are composed of a very heterogeneous population of cells and they all can significantly affect T-cell functions. APCs can regulate T-cell functions through multiple mechanisms, particularly through the action of paracrine factors (cytokines) and through co-stimulatory or inhibitory surface molecules.

that an increasing number of CD8+

86 Molecular Mechanisms of the Aging Process and Rejuvenation

44+

the reduction in the turnover of memory-like CD44+

suggested that the reduced turnover of aged CD44+

44+

44+

44+

The population of CD8+

assumed that a shorter time CD8+

for fast proliferation and old for CD8+

young CD8+

young CD8+

CD8+

**parabiosis**

44+ cells may be mediated by an increase in the number of

memory-like cells is short-lived and quickly proliferating. It is

T-cell recycling relative to naïve CD8+ T-cells is associated

CD8+ cells was found. The authors

cells reflected an inhibitory influence

44+ cells in young

dendritic cells).

cells in the spleen, bone marrow, and thymus of old partners [23]. We hy‐

cells to the old partner, or the proliferation of young cells into the old body

CD8+

memory cell differentiation.

pothesized that these changes may have multiple mechanisms of occurrence: the migration of

through antigen-specific or homeostatic proliferation. However, we cannot assert which of the proposed mechanisms is the key in age-related changes on the basis of the obtained results.

with the higher speed of their homeostatic proliferation [26]. At the same time, in aged mice,

of the aged environment, including APCs [27–29]. The presented data may indicate that the old lymphoid microenvironment can induce changes in the differentiation and functioning of

heterochronic partners may result from the mutual influence of the two environments—young

The obtained data indicate that our hypothesis—that the mechanisms of the immune aging can be mediated by dysfunctions of lymphoid cells and organs (thymus, bone marrow) or insufficient production of some factors (hormones, cytokines, and growth factors)—is un‐ founded. The obtained results showed that the general circulation between partners of different ages, which provides a mutual exchange of hematopoietic stem cells, naïve lympho‐ cytes, hormones, cytokines, and growth factors, does not lead to the restoration of the old immune system functioning. On the contrary, it was found that the common bloodstream from the old to the young partner is able to induce the aging of a young immune system. It is assumed that changes in the proliferation and differentiation of young CD8+ T-cells, which presumably enhanced by their homeostatic proliferation, appear due to the influence of the old lymphoid niches. To test this hypothesis, we used the model of heterochronic cocultivation of lympho‐

**2.3. The study of age-related changes of lymphoid niche cells in the model of heterochronic**

It is well known that peripheral T-cell functions are regulated by multiple extrinsic factors, in particular by the microenvironment of lymphoid organs, where T-cells usually form special zones such as a periarteriolar lymphoid sheath in the spleen. T-cell activities in these zones are regulated by cells of their niche. Lymphoid niche includes fibroblastic reticular cells, which organize T-cell zones and antigen-presenting cells. APCs are important T-cell regulators and include a broad variety of different cell types such as dendritic cells, B-cells, and macrophages. Dendritic cells are professional APCs and regulatory cells, and are able to either induce tolerance or stimulate profound immune response in an organism [30]. Macrophages can also display some DC-like features [31]. APCs are composed of a very heterogeneous population of cells and they all can significantly affect T-cell functions. APCs can regulate T-cell functions

T-cells. We suggested that the rapid growth of the number of CD8+

44+

cytes with cells that form spleen lymphoid niche (macrophages or CD 11c+

The study of DC and macrophage functions is a very difficult task, since in situ these cells are part of an extremely complex structure, forming lymphoid organ stromal microenvironment. It makes it almost impossible to evaluate their native functions in vivo. Therefore, we have created in vitro system to assess the co-stimulatory properties of individual types of lymphoid stromal cells. For investigation, we chose macrophages and DC of the spleen, which are able to influence the antigen-specific and homeostatic proliferation of T-cells. To assess its func‐ tions, we have chosen the model of coculturing APCs with purified T-cells in vitro after stimulation by PHA. To determine the overall effect of heterochronic parabiosis on APCs, we evaluated PHA-mediated T-cell stimulation in the presence of a particular type of APCs either total population of CD11c+ splenic dendritic cells or enriched macrophage population (splenic adherent cells).

T-cells are stimulated via binding of PHA (T-cell superantigen) with T-cell receptor, which results in polyclonal activation of T-cells. The procedure is simple and reliable to determine T-cell antigen-induced proliferation because it mimics the binding of presented antigen with T-cell receptor [32] which does not involve direct artificial activation of intracellular signal cascades.

T-cells were isolated from the total suspension of splenocytes using columns with nylon wool (Polyscience, Inc.) according to the manufacturer's instruction. Flow cytometry analysis of enriched T-cell suspension (stained by anti-CD3-FITC monoclonal antibody (Pickcell Labora‐ tory, the Netherlands) indicated 90–95% purity. Dendritic cells were isolated from the spleen by a CD11c+ selection magnetic separation kit EasySep (Stemcell Technology) according to the manufacturer's instruction. The total population of enriched splenic macrophages was harvested from the splenocytes by adhesion to plastic according to the routine method [23].

Coculturing experiments were carried out to examine the capability of total splenic macro‐ phages and dendritic cells to co-stimulate T-cell proliferation. Coculture experiments included the following combinations: (1) purified splenic T-cells from control young intact mice were cocultured with enriched population of splenic dendritic cells of parabionts, (2) purified splenic T-cells from control young intact mice were cocultured with enriched population of splenic macrophages of parabionts, (3) purified splenic T-cells from parabionts were cocul‐ tured with enriched population of autologous splenic dendritic cells, and (4) purified splenic T-cells from parabionts were cocultured with enriched population of autologous splenic macrophages.

The results of experiment are shown in **Figure 3**. First, we tested PHA-mediated proliferative response of native splenocytes or purified T-cells alone to check the background values, and observed significant differences between young and old isochronic parabionts (**Figure 3A** and **B**, *P*(*t*) < 0.05). The splenocyte proliferation rate in young and old heterochronic parabionts (Yo and Oy) did not differ from the rates of young or old isochronic controls (Yy and Oo). Otherwise, we observed a decrease in proliferative capacity of purified splenic T-cells in the

**Figure 3.** Proliferative response of purified splenic T-cells after PHA stimulation in vitro.Symbol of the experimental groups: Yy, young isochronic animals; Yo, young heretochronic animals; Oy, old heterochronic mice; Oo, old isochron‐ ic mice. CBA/Ca female mice at 1.5 months after the parabiosis initiation was euthanized and subjected to analysis. All pairs with tumors were excluded from the study. **A.** Proliferative activity of native splenocytes. **B.** Proliferative activity of purified splenic T-cells. **C.** Proliferative activity of purified young control T-cells cocultivated with CD11c+ DC from mice of different experimental groups. **D.** Proliferative activity of purified young control T-cells cocultivated with ad‐ herent splenic cells from mice of different experimental groups. **E.** Proliferative activity of purified splenic T-cells co‐ cultivated with autologous CD11c+ DC from experimental mice. **F.** Proliferative activity of purified splenic T-cells cocultivated with autologous adherent splenic cells from experimental mice. \*, *P* (*t*) < 0.05 comparing young isochronic animals; #, *P* (*t*) < 0.05 comparing old intact animals. The mice number in each group is at least 8.

Although the interaction of T-cells usually involves great number of soluble and surface costimulatory molecules, we examined the effect of cocultivation of splenic DCs and macro‐ phages on PHA-induced T-cell activation.

heterochronic parabionts (Yo and Oy) compared to young isochronic control (Yy) (**Figure 3B**,

**Figure 3.** Proliferative response of purified splenic T-cells after PHA stimulation in vitro.Symbol of the experimental groups: Yy, young isochronic animals; Yo, young heretochronic animals; Oy, old heterochronic mice; Oo, old isochron‐ ic mice. CBA/Ca female mice at 1.5 months after the parabiosis initiation was euthanized and subjected to analysis. All pairs with tumors were excluded from the study. **A.** Proliferative activity of native splenocytes. **B.** Proliferative activity of purified splenic T-cells. **C.** Proliferative activity of purified young control T-cells cocultivated with CD11c+

mice of different experimental groups. **D.** Proliferative activity of purified young control T-cells cocultivated with ad‐ herent splenic cells from mice of different experimental groups. **E.** Proliferative activity of purified splenic T-cells co‐

cocultivated with autologous adherent splenic cells from experimental mice. \*, *P* (*t*) < 0.05 comparing young isochronic

animals; #, *P* (*t*) < 0.05 comparing old intact animals. The mice number in each group is at least 8.

DC from experimental mice. **F.** Proliferative activity of purified splenic T-cells

DC from

*P*(*t*) < 0.05).

88 Molecular Mechanisms of the Aging Process and Rejuvenation

cultivated with autologous CD11c+

We used two approaches to assess the activity of spleen dendritic cells and macrophages. The first approach is to assess the age-related APC changes in spleen microenvironment, so we studied the effect of DC and macrophages on the proliferation of intact young T-cells. As shown in **Figure 3C**, we observed significant decrease in the proliferative capacity of enriched population of young intact T-cells, which were cocultivated with CD11c+ DCs of young heterochronic parabionts, but the value was almost same as the cells cocultured with old ones (**Figure 3C**, *P*(*U*) < 0.05). Therefore, we observed the impairment of CD11c+ DC functions for maintaining the proliferation of young T-cells in the group of young heterochronic parabionts. When macrophages were cocultured, we found no similar changes (**Figure 3D**).

Second, we investigated the activation of T-cells by cocultivation with their own dendritic cells or macrophages (autologous mouse) to determine which APC have an inhibitory effect on the activation of T-cells in heterochronic parabionts. As shown in **Figure 3E**, a significant decrease in the proliferation rate of T-cells from old isochronic parabionts cocultured with autologous DCs was observed as compared to the young isochronic ones. Meanwhile, no decrease was observed in the proliferation rate of T-cells cocultured with total splenic dendritic cells from young heterochronic parabionts to the level of young isochronic parabionts. We also analyzed the influence of the total population of splenic adherent cells from animals of different experimental groups on the T-cell proliferation in vitro (**Figure 3F**). The proliferative capacity of T-cells from young heterochronic partners, which were cocultured with their own splenic adherent cells, was found to drop to the level of old animals as compared to young isochronic partners (*P*(*t*) < 0.05). Thus, we suggest that the T-cell proliferative capacity can be strongly impacted by niche-forming cells and their age-related changes can cause T-cell dysfunction. Macrophages could act as T-cell dysfunction inducers in our experimental model of hetero‐ chronic parabiosis.

Finally, we examined key mechanisms of T-cell activation. It has been investigated as compli‐ cated mechanism which includes both the activation process and the apoptosis of activated lymphocytes [32]. We examined the expression of RelA , IkB*α*, and caspase p20 during the activation of T-cells.

**Figure 4** shows the RelA, IkB*α*, and caspase p20 expression of splenic T-cells. The amount of those proteins was determined by using Western blotting method. Coculture with macrophage was used for the experiments because most significant differences were observed. By 2 h of cocultivation of T-cell with macrophages from spleens of autologous donors, a decreased level of RelA and caspase 3 p20 protein in old isochronic parabionts versus the young ones was observed (**Figure 4A** and **C**, respectively, *P*(*U*) < 0.05). In young heterochronic parabionts, Tcell cocultivation with macrophages led to another effect—the RelA level also reduced whereas caspase 3, p20 protein remained unchanged in this group (**Figure 4A** and **C**, respectively, *P*(*U*) <0.05). However, neither significant changes in the IκB*α* level (**Figure 4B**) nor any sign of expression of the full (not active) form of caspase 3 in T-cells was detected (data not presented).

**Figure 4.** Level of RelA, IkBα, and caspase p20 in purified splenic T-cells from parabiotic mice after 2 h of coculturing with splenic macrophage-rich population of adherent cells from animals of the same experimental groups.The total splenic T-cells from parabionts were isolated and 10 × 106 cells were cocultured with previously enriched total splenic macrophages from 10 × 107 splenocytes of mice of the same experimental groups. PHA of 10 μg/ml was added to cell suspensions, and they had been stimulated for 2 h. Then, cells were lysed, and the total cellular extract had been ana‐ lyzed with Western blotting for the main components of T-cell activation signalling: RelA (A), IkBα (B), caspase p20 (C). Data are presented from results of at least three experiments. \*, *P*(*U*) < 0.05 compared to young isochronic para‐ bionts; #, *P*(*U*) < 0.05 compared to young heterochronic parabionts.

Moreover, the experiment of 18 h of cocultivation was performed. By 18 h, we observed equalization of the RelA protein level between T-cells from all experimental groups. No considerable changes in the expression level of the IκB*α* protein in activated T-cells from different experimental groups were marked in this stage of activation. However, the level of the activated form of caspase 3 (p20) was still considerably higher in T-cells from young isochronic and young heterochronic parabionts in comparison with T-cells from either old isochronic or heterochronic parabiotic animals [33].

The experiment focused merely on the key factors of T-cell activation during APC co-stimu‐ lation, and we found out considerable changes in the expression level of several key proteins for T-cell mitogen-induced proliferation. We notice that splenic macrophages can impact Tcell proliferation and mitogen-activated T-cell signaling.

#### **3. Conclusions**

Adaptive immune response most markedly declines with age and main reasons for that are atrophy of the thymus that begins immediately after puberty, and massive changes in the Tcell compartment of lymphoid organs [2]. Defects in T-cell functions greatly affect protective capabilities of an organism so that it becomes more susceptible to different pathogens and cancer [1, 34].

Numerous attempts to rejuvenate the immune system have shown that processes of agerelated changes in T-cell functions are rather complex. Moreover, an interchange between the young immune system and the old one established via the common blood cell circulation induced an age-related decline in the young immune parameters [13].

In the present study, we examined the mechanisms of age-related dysfunction of the immune system using various models of heterochronic chimeras, and analyzing the interaction of various parts of the immune system: hematopoietic stem cells, lymphoid organ microenvir‐ onment, lymphocytes, and systemic factors that circulate in the blood.

Using model of the irradiated chimera, we found that the old hematopoietic stem cells after 3 months residing in lethally irradiated young organism lead to the formation of a smaller amount of CD4+ and CD8+ T-cells in the spleen of young animals than of young HSCs. These data are confirmed by many other studies that reveal a violation of HSCs functioning with age [4, 5]. But the purpose of our study was not to study individual changes of HSCs functions, but the contribution of these changes to the age-related dysfunction of the immune system. According to our findings, changes in the functional properties of old HSCs do not exercise a significant influence on the amplitude of humoral immune response in young animals (**Figure 2**, Yo group). This fact may suggest that the age-related changes in the HSCs cannot cause age-related disorders of the immune system. This supposition is also confirmed by the results, which show that old HSCs lead to a more significant proliferative response of spleno‐ cytes to PHA in old animals when compared with young HSCs (**Figure 1**).

**Figure 4.** Level of RelA, IkBα, and caspase p20 in purified splenic T-cells from parabiotic mice after 2 h of coculturing with splenic macrophage-rich population of adherent cells from animals of the same experimental groups.The total

suspensions, and they had been stimulated for 2 h. Then, cells were lysed, and the total cellular extract had been ana‐ lyzed with Western blotting for the main components of T-cell activation signalling: RelA (A), IkBα (B), caspase p20 (C). Data are presented from results of at least three experiments. \*, *P*(*U*) < 0.05 compared to young isochronic para‐

Moreover, the experiment of 18 h of cocultivation was performed. By 18 h, we observed equalization of the RelA protein level between T-cells from all experimental groups. No considerable changes in the expression level of the IκB*α* protein in activated T-cells from different experimental groups were marked in this stage of activation. However, the level of the activated form of caspase 3 (p20) was still considerably higher in T-cells from young isochronic and young heterochronic parabionts in comparison with T-cells from either old

The experiment focused merely on the key factors of T-cell activation during APC co-stimu‐ lation, and we found out considerable changes in the expression level of several key proteins for T-cell mitogen-induced proliferation. We notice that splenic macrophages can impact T-

Adaptive immune response most markedly declines with age and main reasons for that are atrophy of the thymus that begins immediately after puberty, and massive changes in the Tcell compartment of lymphoid organs [2]. Defects in T-cell functions greatly affect protective capabilities of an organism so that it becomes more susceptible to different pathogens and

Numerous attempts to rejuvenate the immune system have shown that processes of agerelated changes in T-cell functions are rather complex. Moreover, an interchange between the young immune system and the old one established via the common blood cell circulation

induced an age-related decline in the young immune parameters [13].

splenocytes of mice of the same experimental groups. PHA of 10 μg/ml was added to cell

cells were cocultured with previously enriched total splenic

splenic T-cells from parabionts were isolated and 10 × 106

90 Molecular Mechanisms of the Aging Process and Rejuvenation

bionts; #, *P*(*U*) < 0.05 compared to young heterochronic parabionts.

isochronic or heterochronic parabiotic animals [33].

cell proliferation and mitogen-activated T-cell signaling.

macrophages from 10 × 107

**3. Conclusions**

cancer [1, 34].

Next, we used another model—heterochronic parabiosis. This model is characterized by a common blood circulation between animals of different ages, allowing them to share not only stem cells but also blood lymphocytes and circulated growth factors and hormones. But instead of the regeneration of the immune system in old partner, we observed the progressive reduction of the most immunological parameters in young ones in this model.

Accelerated aging of the immune system in young heterochronic partners gave us a unique opportunity in a short time to study the trigger mechanism of these age-related changes. It was determined that the first change appeared within 3 weeks after surgery is the doubled number of CD8+ 44+ memory-like cells in the spleen of young heterochronic partners that reached a level in old mice. Increasing the number of these cells may indicate that there is one possible mechanism of age-related dysfunction of the immune system in heterochronic parabiosis. Since the differentiation of CD8+ 44+ T-cells is provided by their homeostatic proliferation in the peripheral lymphoid organs, we assume that these changes can occur as a result of enhancing their homeostatic proliferation under the influence of old lymphoid niches.

It has been well known that the functioning of T-cells greatly depends on their niche, and APCs being its most essential component. They include B-cells, macrophages, and dendritic cells. The last is considered as the most potent antigen-presenting cell type [31]. A role of APCs in T-cell functioning is hardly to be overestimated—they regulate activation and differentiation of T-cells, shape the TCR repertoire, and assist in the cessation of unimportant immune response. Interactions of APCs with T-cells include TCR-major histocompatibility complex coupling, a contact through an array of surface molecules (adhesion, co-stimulatory, and coinhibitory molecules), and expression of multiple soluble molecules (interleukins and cyto‐ kines).

To determine the effect of heterochronic parabiosis on APC function, we have chosen a model of PHA-mediated T-cell stimulation in the presence of total population of either CD11c+ splenic dendritic cells or macrophages. During this test, T-cells are stimulated via binding of PHA. This way of T-cell stimulation is simple, reliable, and physiological instrument of measuring T-cell antigen-induced proliferation because it mimics the binding of antigen with T-cell receptor [31].

We observed the impairment of CD11c+ DCs for maintaining the proliferation of young T-cells in the group of young heterochronic parabionts (**Figure 4C**).

It has been shown that the ability of splenic CD11c+ dendritic cells to affect the proliferation of autologous T-cells varies slightly between young heterochronic and isochronic parabionts. But these changes are more evident in the fast-renewing population of splenic macrophages. We demonstrated decreased proliferative response of T-cells from young heterochronic parabionts when they were stimulated in vitro in the presence of macrophage-rich population of autolo‐ gous splenocytes. Also, during stimulation in vitro in the presence of autologous macrophages, T-cells from young heterochronic parabionts had a lower expression of NFκB p65 on early stages of stimulation (2 h) and a higher expression of IκBα on later stages (18 h) of stimulation when compared with T-cells from young isochronic parabionts. Observed data indicate the induction of negative changes in functions of macrophage-rich population of splenocytes during heterochronic parabiosis [33].

Summarizing, immune system aging has an extremely complex mechanism, the implementa‐ tion of which involved various cells, organs, and blood factors. Age-related changes give rise to changes in the properties of hematopoietic stem cells, disruption of migration routes, and differentiation mechanisms of immunocompetent T-cells, which ultimately leads to the disruption of T-cell subpopulations of lymphoid organs and their dysfunctions. Attempts to replace the old cells by young ones do not lead to the rejuvenation of the immune system. On the contrary, all the young hematopoietic stem or immune cells that were differentiated in the old organism very quickly become "old" both in its phenotype and on its functional charac‐ teristics. One of the main reasons for these changes is the microenvironment of lymphoid organs, where there is a differentiation and proliferation of HSCs and naïve T-cells. DCs, macrophages, fibroblasts, etc. are those cells which form the stroma of lymphoid organs (or lymphoid niche), and have a significant impact on the differentiation of HSCs or naïve T-cells, such as via the co-stimulatory molecules on the cell surface, as via cytokine production. And the development of approaches to the correction of age-related changes of these cell types, which form niche lymphoid organs, may help restore the age-related immune system dys‐ functions in the future.

#### **Acknowledgements**

The authors are grateful to the Institute of Biology of Aging (Moscow, Russia) for supporting this work.

#### **Author details**

dendritic cells or macrophages. During this test, T-cells are stimulated via binding of PHA. This way of T-cell stimulation is simple, reliable, and physiological instrument of measuring T-cell antigen-induced proliferation because it mimics the binding of antigen with T-cell

It has been shown that the ability of splenic CD11c+ dendritic cells to affect the proliferation of autologous T-cells varies slightly between young heterochronic and isochronic parabionts. But these changes are more evident in the fast-renewing population of splenic macrophages. We demonstrated decreased proliferative response of T-cells from young heterochronic parabionts when they were stimulated in vitro in the presence of macrophage-rich population of autolo‐ gous splenocytes. Also, during stimulation in vitro in the presence of autologous macrophages, T-cells from young heterochronic parabionts had a lower expression of NFκB p65 on early stages of stimulation (2 h) and a higher expression of IκBα on later stages (18 h) of stimulation when compared with T-cells from young isochronic parabionts. Observed data indicate the induction of negative changes in functions of macrophage-rich population of splenocytes

Summarizing, immune system aging has an extremely complex mechanism, the implementa‐ tion of which involved various cells, organs, and blood factors. Age-related changes give rise to changes in the properties of hematopoietic stem cells, disruption of migration routes, and differentiation mechanisms of immunocompetent T-cells, which ultimately leads to the disruption of T-cell subpopulations of lymphoid organs and their dysfunctions. Attempts to replace the old cells by young ones do not lead to the rejuvenation of the immune system. On the contrary, all the young hematopoietic stem or immune cells that were differentiated in the old organism very quickly become "old" both in its phenotype and on its functional charac‐ teristics. One of the main reasons for these changes is the microenvironment of lymphoid organs, where there is a differentiation and proliferation of HSCs and naïve T-cells. DCs, macrophages, fibroblasts, etc. are those cells which form the stroma of lymphoid organs (or lymphoid niche), and have a significant impact on the differentiation of HSCs or naïve T-cells, such as via the co-stimulatory molecules on the cell surface, as via cytokine production. And the development of approaches to the correction of age-related changes of these cell types, which form niche lymphoid organs, may help restore the age-related immune system dys‐

The authors are grateful to the Institute of Biology of Aging (Moscow, Russia) for supporting

DCs for maintaining the proliferation of young T-cells

receptor [31].

We observed the impairment of CD11c+

92 Molecular Mechanisms of the Aging Process and Rejuvenation

during heterochronic parabiosis [33].

functions in the future.

**Acknowledgements**

this work.

in the group of young heterochronic parabionts (**Figure 4C**).

Iryna Pishel1\* and Gennadij Butenko2

\*Address all correspondence to<EMAIL_ADDRESS>
1 Leader Scientific Researcher, Laboratory of Pathophysiology and Immunology, D. F. Chebotaryov State Institute of Gerontology NAMS of Ukraine, Kyiv, Ukraine

2 Head of the Laboratory of Pathophysiology and Immunology, D. F. Chebotaryov State In‐ stitute of Gerontology NAMS of Ukraine, Kyiv, Ukraine

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## **New Perspectives in the Diagnosis of Mild Cognitive Impairment and Alzheimer's Disease: Novel Uses of Biomarkers**

Judit Subirana-Mirete

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20150302.14

96 Molecular Mechanisms of the Aging Process and Rejuvenation

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64322

#### **Abstract**

Criteria for the diagnosis of Alzheimer's disease were established in 1984 by the National Institute of Neurological and Communicative Disorders and Stroke (NINCDS) and the Alzheimer's Disease and Related Disorders Association (ADRDA). Common‐ ly used since their implementation, these criteria are becoming obsolete for everyday practice, and a review is being claimed. Three groups of experts consisting of re‐ nowned experts from the National Institute on Aging (NIA) and the Alzheimer's Association proposed a set of recommendations to modify the criteria in this field of research. Two notable differences from the initial criteria were included: the incorpo‐ ration of biomarkers and the formalization of different disease stages in the diagnos‐ tic criteria. From now on, mild cognitive impairment is incorporated in the diagnosis as another stage of dementia. However, the new criteria are still under revision and are currently of use for research purposes with the aim to get the definitive modification for the clinical criteria. This chapter presents the main developments in research concerning Alzheimer's disease and mild cognitive impairment to define these new research criteria.

**Keywords:** Alzheimer's disease, mild cognitive impairment, biomarkers, neuropsy‐ chology, dementia

#### **1. Introduction**

The concept of dementia makes us instantly think about a set of characteristic symptoms and the resulting repercussions for patients and their families. The objective of this chapter is to

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

update our ideas about Alzheimer's disease and get to know the beginning and evolution of the construct of mild cognitive impairment, as well as the new advances in genetics and biomark‐ ers that, in not such a distant future, will be of common use in the diagnosis of cognitive impairment.

The Greeks were the first to study mental disorders from a scientific point of view by separating the study of mind from religion. The existence of dementia has been known since the times of Hippocrates (460–370 BC), although throughout history, it has been given different names: paranoia, idiocy, senility, senile psychosis, and so on. But it will not be until 1906 that Alois Alzheimer first described the disease that would be named after him and which has made history worldwide [1].

From Dr. Alois Alzheimer's first patient, Auguste D., until the current description and definition of the disease, more than a century has passed and many research studies have been carried out on it. Initially, it was considered to be a condition particular to young patients, and so it was given the name of presenile dementia. However, the progressive increase in life expectancy left uncovered that the symptoms present in Auguste D. were also found in older population, thus leading to a new approach to the disease. But it was not until the 1970s that the disease described by Alzheimer started to be a focus of attention. The real story of the concept of dementia and Alzheimer's disease can be consulted in different texts of reference [1–3].

Recently, knowledge on dementia in general and on Alzheimer's disease in particular has been remarkably expanded. The diagnostic process for Alzheimer's disease has benefitted from widely accepted consensus protocols [4]. Nevertheless, the clinical heterogeneity of this disease (age of onset, type of impairment, or the disease's progression pace) makes diagno‐ sis extremely difficult.

#### **2. Diagnosis of Alzheimer's disease**

Alzheimer's disease is a neurodegenerative condition of insidious onset and progressive evolution, characterized by loss of memory and other cognitive functions, and by a set of noncognitive symptoms, among which depressive or psychotic-related symptoms and behaviour disorders stand out. Among the main neuropsychological features of this disease, we can point at the progressive impairment of memory and language, the decline in visuo‐ spatial and motor capacities, and the disruption of executive functions such as the capacity for abstraction and reasoning [5, 6].

The criteria for the clinical diagnosis of Alzheimer's disease were defined in 1984 by the National Institute of Neurological and Communicative Disorders (NINCD) and the Alzheim‐ er's Disease and Related-Disorders Association (ADRDA). These criteria are based on the idea that Alzheimer's disease is a single clinicopathological entity [7], thus advocating that Alzheimer's disease always has a close correlation between clinical symptoms and their pathological basis; in most cases, it was thought that the typical anatomopathological presen‐ tation already described in 1910 and based on the presence of senile plaques, neurofibrillary tangles and cerebral arteriosclerotic changes [8] would be found at autopsy. Following this idea, the basic lines for the development of the disease were, in 1984, as follows:

Thus, 1984 criteria required the presence of cognitive impairment and dementia to be confirmed by neuropsychological assessment in order to establish the diagnosis of possible or probable Alzheimer's disease, although histopathological confirmation was still needed for a final diagnosis (via autopsy or biopsy) (see **Table 1**).

Source: Adapted from Carrasco [21].

update our ideas about Alzheimer's disease and get to know the beginning and evolution of the construct of mild cognitive impairment, as well as the new advances in genetics and biomark‐ ers that, in not such a distant future, will be of common use in the diagnosis of cognitive

The Greeks were the first to study mental disorders from a scientific point of view by separating the study of mind from religion. The existence of dementia has been known since the times of Hippocrates (460–370 BC), although throughout history, it has been given different names: paranoia, idiocy, senility, senile psychosis, and so on. But it will not be until 1906 that Alois Alzheimer first described the disease that would be named after him and which has made

From Dr. Alois Alzheimer's first patient, Auguste D., until the current description and definition of the disease, more than a century has passed and many research studies have been carried out on it. Initially, it was considered to be a condition particular to young patients, and so it was given the name of presenile dementia. However, the progressive increase in life expectancy left uncovered that the symptoms present in Auguste D. were also found in older population, thus leading to a new approach to the disease. But it was not until the 1970s that the disease described by Alzheimer started to be a focus of attention. The real story of the concept of dementia and Alzheimer's disease can be consulted in different texts of reference

Recently, knowledge on dementia in general and on Alzheimer's disease in particular has been remarkably expanded. The diagnostic process for Alzheimer's disease has benefitted from widely accepted consensus protocols [4]. Nevertheless, the clinical heterogeneity of this disease (age of onset, type of impairment, or the disease's progression pace) makes diagno‐

Alzheimer's disease is a neurodegenerative condition of insidious onset and progressive evolution, characterized by loss of memory and other cognitive functions, and by a set of noncognitive symptoms, among which depressive or psychotic-related symptoms and behaviour disorders stand out. Among the main neuropsychological features of this disease, we can point at the progressive impairment of memory and language, the decline in visuo‐ spatial and motor capacities, and the disruption of executive functions such as the capacity for

The criteria for the clinical diagnosis of Alzheimer's disease were defined in 1984 by the National Institute of Neurological and Communicative Disorders (NINCD) and the Alzheim‐ er's Disease and Related-Disorders Association (ADRDA). These criteria are based on the idea that Alzheimer's disease is a single clinicopathological entity [7], thus advocating that Alzheimer's disease always has a close correlation between clinical symptoms and their pathological basis; in most cases, it was thought that the typical anatomopathological presen‐

impairment.

[1–3].

history worldwide [1].

98 Molecular Mechanisms of the Aging Process and Rejuvenation

sis extremely difficult.

**2. Diagnosis of Alzheimer's disease**

abstraction and reasoning [5, 6].

**Table 1.** Criteria for the diagnosis of Alzheimer's disease.

Some aspects of these criteria have been set aside by new research and daily clinical practice has also pointed at ideas to be revised. The pathological histology present in Alzheimer's disease can also be found in a wide set of alternative clinical conditions [9, 10], from patients without cognitive symptoms to patients with mild cognitive impairment (MCI) or other types of impairment. Therefore, the initial concept of Alzheimer's disease has to be less restrictive than that developed in 1984.

The capacity to recognize some clinical symptoms in other disorders with a similar develop‐ ment as in Alzheimer's disease was limited two decades ago, thus resulting in diagnostic confusions. In this respect, for example, reversible systemic disorders such as vitamin B12 deficiency, which may have similar symptoms to a dementia, were not taken into consideration [11]. Although frontotemporal dementia was considered to be an entity, others such as Lewy body dementia or vascular dementia were not taken into account [12]. Likewise, the concept of aphasia linked to neurodegenerative disorders, despite being described some years before the NINCDS-ADRDA criteria [13], was not fully developed until two decades later [14].

The implication that memory impairment must always be considered as the primary cognitive deficit for the diagnosis of Alzheimer's disease is also being put into doubt, as clinical experi‐ ence has showed that Alzheimer's disease may be developed in a nonamnesic way [15]. Clinical experience has also pointed at the need to revise cut-off points by age for the diagnosis, as agerelated pathologies such as dementia are more and more frequent due to the overageing of population. Finally, the inclusion of new results from neuroimaging exploration and biomark‐ er clinical analysis in the new criteria will allow us to get to know the individual characteristics of every cognitive impairment from an integral approach of the same construct.

Since the establishment of the NINCDS-ADRDA criteria 32 years ago, it has been proved that Alzheimer's disease underlying pathology and clinical symptoms are not always present concurrently, thus dismissing one of the main assumptions of the 1984 diagnostic criteria. Research has evolved remarkably since then and, among other events, it has been identified, for example, that in the absence of any apparent symptom, there can be a wide pathological presentation (particularly of amyloid plaques) [16, 17].

Knowledge about the neuropathology of Alzheimer's disease has been expanded in the last 25 years of the twentieth century; new sets of diagnostic criteria to establish a diagnosis of this disease from its neuropathological basis have been developed and applied more or less successfully. Among others, we have to consider those by the National Institute on Aging (NIA) [18], those by the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) [19], and those by the NIA-Reagan Institute [20]. There are no basic differences among these three classifications; they all stand out the need to carry out an inclusive diagnosis, establish the priority for the cognitive diagnosis—with special emphasis on memory—and consider other noncognitive or psychiatric symptoms to be "accessory" [21]. These approaches present two main problems: first, they downplay cognitive symptoms, as they only focus on amnesic manifestations, with the other cognitive functions and psychopathological impairments being left in the background; second, they are limited by the inability to categorize certain symptoms present in this kind of dementia [22]. Nevertheless, there are two main research aspects that have evolved from the very first description of this disease: using biomarkers and formally establishing different stages for the disease. Literature on mild cognitive impairment has exponentially increased since the 1990s in order to document the gradual impairment of cognitive functions preceding the point when there is a significant interference with activities of daily living [23]. As previously mentioned, the 1984 criteria did not consider cognitive impairment that does not reach the dementia threshold, thus passing over that the Alzheimer's disease underlying pathology slowly develops for years—or even decades—before there is a clinical manifestation of the disease.

#### **3. Mild cognitive impairment**

Some aspects of these criteria have been set aside by new research and daily clinical practice has also pointed at ideas to be revised. The pathological histology present in Alzheimer's disease can also be found in a wide set of alternative clinical conditions [9, 10], from patients without cognitive symptoms to patients with mild cognitive impairment (MCI) or other types of impairment. Therefore, the initial concept of Alzheimer's disease has to be less restrictive

The capacity to recognize some clinical symptoms in other disorders with a similar develop‐ ment as in Alzheimer's disease was limited two decades ago, thus resulting in diagnostic confusions. In this respect, for example, reversible systemic disorders such as vitamin B12 deficiency, which may have similar symptoms to a dementia, were not taken into consideration [11]. Although frontotemporal dementia was considered to be an entity, others such as Lewy body dementia or vascular dementia were not taken into account [12]. Likewise, the concept of aphasia linked to neurodegenerative disorders, despite being described some years before the NINCDS-ADRDA criteria [13], was not fully developed until two decades later [14].

The implication that memory impairment must always be considered as the primary cognitive deficit for the diagnosis of Alzheimer's disease is also being put into doubt, as clinical experi‐ ence has showed that Alzheimer's disease may be developed in a nonamnesic way [15]. Clinical experience has also pointed at the need to revise cut-off points by age for the diagnosis, as agerelated pathologies such as dementia are more and more frequent due to the overageing of population. Finally, the inclusion of new results from neuroimaging exploration and biomark‐ er clinical analysis in the new criteria will allow us to get to know the individual characteristics

Since the establishment of the NINCDS-ADRDA criteria 32 years ago, it has been proved that Alzheimer's disease underlying pathology and clinical symptoms are not always present concurrently, thus dismissing one of the main assumptions of the 1984 diagnostic criteria. Research has evolved remarkably since then and, among other events, it has been identified, for example, that in the absence of any apparent symptom, there can be a wide pathological

Knowledge about the neuropathology of Alzheimer's disease has been expanded in the last 25 years of the twentieth century; new sets of diagnostic criteria to establish a diagnosis of this disease from its neuropathological basis have been developed and applied more or less successfully. Among others, we have to consider those by the National Institute on Aging (NIA) [18], those by the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) [19], and those by the NIA-Reagan Institute [20]. There are no basic differences among these three classifications; they all stand out the need to carry out an inclusive diagnosis, establish the priority for the cognitive diagnosis—with special emphasis on memory—and consider other noncognitive or psychiatric symptoms to be "accessory" [21]. These approaches present two main problems: first, they downplay cognitive symptoms, as they only focus on amnesic manifestations, with the other cognitive functions and psychopathological impairments being left in the background; second, they are limited by the inability to categorize certain symptoms present in this kind of dementia [22]. Nevertheless, there are two main research aspects that have evolved from the very first description of this disease: using biomarkers and formally

of every cognitive impairment from an integral approach of the same construct.

presentation (particularly of amyloid plaques) [16, 17].

than that developed in 1984.

100 Molecular Mechanisms of the Aging Process and Rejuvenation

Different nosological entities have been defined in order to characterize cognitive impairment processes that represent an intermediate stage between cognitive decline observed in the ageing process and changes that meet the criteria for the diagnosis of dementia [24]. The most used concept is that of "mild cognitive impairment" (MCI) coined by Petersen et al. [25], although initially described by Flicker et al. [26] according to Reisberg's Global Deterioration Scale [27].

MCI is a syndrome that shows up as a cognitive dysfunction greater than expected for the patient's age and cultural and educational level, that develops without major interferences in activities of daily living and does not meet criteria for dementia [25, 28]. The main diagnostic difference between Alzheimer's disease and mild cognitive impairment lies in the degree of interference in the patient's capacity to develop daily work or regular tasks. This is, unavoid‐ ably, a determination to be carried out by an expert on the basis of individual circumstances and the description of daily routines obtained by both the patient and a close informant. For a further development of this differential diagnosis, please see Albert et al. [29].

The definition in Petersen [21] describes MCI as a degenerative process that precedes dementia —on the basis of the *cognitive continuum* construct (**Figure 1**), where the main deficit concerns memory and the other cognitive functions seem to be stable. Later studies have expanded the MCI construct by describing different subtypes not only limited to amnestic symptoms [30].

Nelson and O'Connor [31] described, through postmortem histopathological studies with MCI patients, that a wide majority of cases evolved to a process of dementia, although a small percentage did not. These results put into question the assumption that MCI may always be considered as an initial stage of Alzheimer's disease.

As we get closer to the definition of the earliest stages of cognitive impairment, the dissociation between the connotations of the concept of early Alzheimer's disease and MCI is more obvious. It is becoming clearer and clearer that both Alzheimer's disease pathophysiological process and symptoms are better explained as a continuum (linking both Alzheimer's disease and MCI at the same time), as these processes can concurrently evolve and even overlap in time [32]. Hence the need to revise both constructs further.

**Figure 1.** Model of cognitive continuum. Adapted from Sperling et al. [32].

Although some agreement has been reached in the last few years, the current MCI diagnosis is complex as there are no precise and standardized criteria with general consensus. As main characteristics, we can stand out the complaints about memory loss from the beginning of the process, reported either by an informant or by the patients themselves. Patients often report other symptoms, such as difficulties to find needed words, loss of personal objects, disorien‐ tation, or loss of continuity in usual tasks, for example, in a conversation [31].

There have been attempts to formalize these clinical observations as diagnostic criteria. We can stand out those by the Study Group on Dementia and Behavioral Disorders of the Spanish Society of Neurology [33] or those by Mayo Clinic [25] that correspond to the amnestic subtype only. Also worth highlighting are those by the International Psychogeriatric Association (IPA) [34], described in **Table 2**.

Despite the efforts to agree on diagnostic criteria for MCI, there are still some difficulties deriving, in many cases, from conceptual limitations of the nosological entity itself [24]. Since the Montreal Consensus [35], criteria became more flexible, with the inclusion, for instance, not only of patients in a cognitive normality dementia transition stage but also very prevalent intermediate conditions secondary to other aetiologies (e.g., vascular dementia) or other conditions, also very frequent, such as those secondary to mood disorders [36].

One of the facts that has always been an object of attention is the relationship between MCI and dementia, in particular, Alzheimer's disease [30, 37]. A recently published study [38] reports the prevalence for mild cognitive impairment between 3 and 42% depending on the construct. We should remember now that, before Petersen et al.'s definition of MCI [25], other constructs had been considered such as age-associated memory impairment (AAMI) [27] or cognitive impairment no dementia (CIND) [39]. In particular, according to Ward's study, AAMI prevalence is between 3.6 and 38.4%; the prevalence for CIND ranges between a bit more than 5 and 35.9%, whereas studies using the concept of MCI (as described by Petersen) show population prevalence between 3 and 42%. Moreover, if we consider that MCI patients have a three times higher risk to develop Alzheimer's disease in a period of 4.5 years after diagnosis [40], the need for some criteria that are agreed upon, global and useful for the diagnosis of cognitive impairment in all of its stages, from onset to more serious stages, becomes particularly important.

**Diagnostic criteria according to the International Psychogeriatric Association (IPA) and World Health Organization (WHO)**

**Table 2.** IPA criteria.

**Figure 1.** Model of cognitive continuum. Adapted from Sperling et al. [32].

102 Molecular Mechanisms of the Aging Process and Rejuvenation

[34], described in **Table 2**.

Although some agreement has been reached in the last few years, the current MCI diagnosis is complex as there are no precise and standardized criteria with general consensus. As main characteristics, we can stand out the complaints about memory loss from the beginning of the process, reported either by an informant or by the patients themselves. Patients often report other symptoms, such as difficulties to find needed words, loss of personal objects, disorien‐

There have been attempts to formalize these clinical observations as diagnostic criteria. We can stand out those by the Study Group on Dementia and Behavioral Disorders of the Spanish Society of Neurology [33] or those by Mayo Clinic [25] that correspond to the amnestic subtype only. Also worth highlighting are those by the International Psychogeriatric Association (IPA)

Despite the efforts to agree on diagnostic criteria for MCI, there are still some difficulties deriving, in many cases, from conceptual limitations of the nosological entity itself [24]. Since the Montreal Consensus [35], criteria became more flexible, with the inclusion, for instance, not only of patients in a cognitive normality dementia transition stage but also very prevalent intermediate conditions secondary to other aetiologies (e.g., vascular dementia) or other

One of the facts that has always been an object of attention is the relationship between MCI and dementia, in particular, Alzheimer's disease [30, 37]. A recently published study [38] reports the prevalence for mild cognitive impairment between 3 and 42% depending on the construct. We should remember now that, before Petersen et al.'s definition of MCI [25], other constructs had been considered such as age-associated memory impairment (AAMI) [27] or cognitive impairment no dementia (CIND) [39]. In particular, according to Ward's study,

tation, or loss of continuity in usual tasks, for example, in a conversation [31].

conditions, also very frequent, such as those secondary to mood disorders [36].

Since 2009 there has been a consensus among the main research centres, that diagnostic criteria for Alzheimer's disease, as well as those for dementia and mild cognitive impairment, should be revised. For this reason, the National Institute on Aging (NIA), together with the Alzheim‐ er's Association, sponsored a deep revision of criteria, thus establishing a revision of old criteria by three independent groups of experts. The first group undertook to establish and describe diagnostic criteria for dementia and Alzheimer's disease; the second group focused on the symptomatic stage prior to Alzheimer-like dementia; finally, the third group dealt with the asymptomatic stage prior to dementia and mild cognitive impairment. The recommendations of the three groups were presented at the 2010 International Conference on Alzheimer's Disease and later published [41]. This new proposal of criteria has integrated research on dementia, MCI, and Alzheimer's disease carried out in the last 25 years of the twentieth century. In particular, the inclusion of genetic breakthroughs, biomarkers, and final formali‐ zation of the different stages in the development of dementia are worth standing out.

#### **4. New advances in research**

The role of genetics in the diagnosis of the different types of cognitive impairment is more and more active and important. At present, there is no doubt about the existence of genetic risk factors to develop Alzheimer's disease, as some genes have been identified that are not only responsible for the genetically pure forms of the disease but also some sporadic and late-onset forms [42].

Although the most important risk factor related to Alzheimer's disease is the ageing process of the patient, the second risk factor is the family history of the disease. Thus, Alzheimer's disease is a complex pathology with a clear genetic component. Up to now, three genes have been found to be responsible for early onset familial Alzheimer's disease: the gene for amyloid beta precursor protein (APP), the gene for presenilin-1 (PS1), and the gene for presenilin-2 (PS2). Taking this into account, if the predominant autosomal form of Alzheimer's disease is present in a patient with MCI, then this MCI is more likely to be a prodrome of early onset Alzheimer's disease [43].

But not only is early onset Alzheimer's disease linked to genetic influence. Approximately, 40% of diagnosed individuals have a family history of the disease, and epidemiological studies show that the risk of developing the disease if a first-degree relative already has it is between two and three times higher than that of general population [44]. The genetic component of lateonset Alzheimer's disease has been targeted in many studies, but at the moment only a genetic component, APOE, has been considered as a risk factor associated with the disease [43]. Thus, up to now, the presence of one or two **ε**4 alleles in the gene for apolipoprotein E (APOE) has been the only genetic variation widely accepted as a risk factor to develop late-onset Alzheim‐ er's disease, whereas the presence of the **ε**2 allele would have the opposite effect [45]. Evidence suggests, then, that an individual meeting clinical, cognitive, and etiological criteria for MCI and carrier of the **ε**4 allele of the APOE gene is more likely to progress towards developing Alzheimer's disease, eventually, than any other individual without this genetic characteristic.

Genetic research has also allowed us to know that the molecular mechanisms that start Alzheimer's disease, both clinically and pathologically, correspond to a metabolic disorder of amyloid beta (Aβ) [46]. A key point is the concept according to which some usually soluble neuronal proteins can misfold and aggregate, for instance, in neurofibrillary tangles, thus producing high levels of cell cytotoxicity [47]. Recent data suggest that although familial Alzheimer's disease is characterized by Aβ overproduction, sporadic late-onset forms are characterized by a decrease in Aβ recycling capacity. And Aβ traffic is controlled by APOE, so the genetic data available at the moment overwhelmingly point at the amyloid cascade hypothesis as the starter of the cognitive impairment process [48].

To facilitate the theoretical discussion of the MCI construct as a preclinical stage of Alzheimer's disease, the group revising the criteria [32] proposed a theoretical model to explain the relationship between cognitive decline and the basic pathophysiology of Alzheimer's disease (see **Figure 2**). As will be seen later, genetic factors are not the only determinants in the development and progression of mild cognitive impairment, as other risk factors, such as vascular factors or the history of personal development, as well as environmental factors and cognitive reserve, which can positively influence the development of cognitive symptoms of decline, also have to be considered.

**4. New advances in research**

104 Molecular Mechanisms of the Aging Process and Rejuvenation

forms [42].

Alzheimer's disease [43].

The role of genetics in the diagnosis of the different types of cognitive impairment is more and more active and important. At present, there is no doubt about the existence of genetic risk factors to develop Alzheimer's disease, as some genes have been identified that are not only responsible for the genetically pure forms of the disease but also some sporadic and late-onset

Although the most important risk factor related to Alzheimer's disease is the ageing process of the patient, the second risk factor is the family history of the disease. Thus, Alzheimer's disease is a complex pathology with a clear genetic component. Up to now, three genes have been found to be responsible for early onset familial Alzheimer's disease: the gene for amyloid beta precursor protein (APP), the gene for presenilin-1 (PS1), and the gene for presenilin-2 (PS2). Taking this into account, if the predominant autosomal form of Alzheimer's disease is present in a patient with MCI, then this MCI is more likely to be a prodrome of early onset

But not only is early onset Alzheimer's disease linked to genetic influence. Approximately, 40% of diagnosed individuals have a family history of the disease, and epidemiological studies show that the risk of developing the disease if a first-degree relative already has it is between two and three times higher than that of general population [44]. The genetic component of lateonset Alzheimer's disease has been targeted in many studies, but at the moment only a genetic component, APOE, has been considered as a risk factor associated with the disease [43]. Thus, up to now, the presence of one or two **ε**4 alleles in the gene for apolipoprotein E (APOE) has been the only genetic variation widely accepted as a risk factor to develop late-onset Alzheim‐ er's disease, whereas the presence of the **ε**2 allele would have the opposite effect [45]. Evidence suggests, then, that an individual meeting clinical, cognitive, and etiological criteria for MCI and carrier of the **ε**4 allele of the APOE gene is more likely to progress towards developing Alzheimer's disease, eventually, than any other individual without this genetic characteristic.

Genetic research has also allowed us to know that the molecular mechanisms that start Alzheimer's disease, both clinically and pathologically, correspond to a metabolic disorder of amyloid beta (Aβ) [46]. A key point is the concept according to which some usually soluble neuronal proteins can misfold and aggregate, for instance, in neurofibrillary tangles, thus producing high levels of cell cytotoxicity [47]. Recent data suggest that although familial Alzheimer's disease is characterized by Aβ overproduction, sporadic late-onset forms are characterized by a decrease in Aβ recycling capacity. And Aβ traffic is controlled by APOE, so the genetic data available at the moment overwhelmingly point at the amyloid cascade

To facilitate the theoretical discussion of the MCI construct as a preclinical stage of Alzheimer's disease, the group revising the criteria [32] proposed a theoretical model to explain the relationship between cognitive decline and the basic pathophysiology of Alzheimer's disease (see **Figure 2**). As will be seen later, genetic factors are not the only determinants in the development and progression of mild cognitive impairment, as other risk factors, such as

hypothesis as the starter of the cognitive impairment process [48].

**Figure 2.** Relationship between cognitive decline and the basic pathophysiology of Alzheimer's disease. Based on Sperling et al. [32].

Another remarkable difference between the 1984 criteria and those revised in 2011 is the incorporation of biomarkers to diagnose cognitive impairment. Biomarkers are physiological, biochemical, or anatomic parameters that are measured in vivo and that reflect specific characteristics of pathophysiological processes related to a disease; in this case, MCI or Alzheimer's disease. It is important to incorporate new knowledge on biomarkers into the diagnostic framework of these diseases, as, on the one hand, they provide us with greater support when establishing the etiological bases of the cognitive decline process and thus allow us to better choose the intervention to follow when there are effective specific treatments; on the other hand, they allow us to determine the probability of the cognitive and functional progression of impairment towards a more serious stage of MCI or towards dementia, as well as the possibility that this progression develops in a defined period of time [29].

There are many biomarkers. Nevertheless, after a long and thorough study of the main markers, the study groups selected only two categories of biomarkers to be included in their recommendations. These are the biomarkers related to Aβ and the biomarkers reflecting neuronal injury [29].

The amyloid plaques (or senile plaques) are one of the most characteristic features of Alz‐ heimer's disease and, therefore, those biomarkers that can detect and quantify Aβ protein or tau protein accumulated in brain tissue are vitally important for the pathological diagnosis of the disease and its precursors [49]. Aβ deposition markers include both measurements of the level of Aβ42 in cerebrospinal fluid (CSF) as evidence of deposition via positron emission tomography (PET) imaging by using a variety of specific binding agents [50]. Tau protein accumulation markers can be obtained through measurements via CSF.

Among neuronal injury markers, there is a series of structural and functional measures, described in **Table 3**, together with Aβ biomarkers.

*Abbreviations*: CSF, cerebrospinal fluid; PET, positron emission tomography; FDG, fluorodeoxyglucose; SPECT, single photon emission tomography; fMRI, functional magnetic resonance imaging; BOLD, blood oxygen level-dependent; MR, magnetic resonance. Adapted from Albert et al. [29].

**Table 3.** Biomarkers for clinical diagnosis.

Among the potential uses of biomarkers, there is the identification of people in the preclinical stages of the disease or the reduction in the heterogeneity of the disease in clinical trials. However, not all biomarkers are valid as indirect assessment criteria, as they can be difficult to validate and require different levels of validation depending on their expected use. In this respect, although the presence of high levels of tau protein is clearly associated with the pathophysiological process of Alzheimer's disease, changes in tau and phosphorylated-tau (p-Tau) are not exclusive to Alzheimer's disease and can also reflect more general neuronal and synaptic damage. The same can be said of Aβ depositions, as they are not exclusive to Alzheimer's disease. This is one of the main limitations of these biomarkers [51]. No conclusive determination has been reached either concerning whether the quantitative measure of these biomarkers provides us with more information than the dichotomous assessment (presence/ absence; positive/negative). Nevertheless, the combination of both types of measures points at the higher probability that the pathophysiological process of the disease is the etiological base of underlying changes [29].

One of the main drawbacks in Alzheimer's disease research is that the symptoms of the disease appear after significant neuronal loss. The objective of current research with biomarkers is to manage to prevent this loss before the final emergence of symptoms, in order to develop really efficient treatments against this disease, as current medication can only provide patients with short-term improvements of their cognitive function [52].

### **5. New criteria for research on mild cognitive impairment and Alzheimer's disease**

The amyloid plaques (or senile plaques) are one of the most characteristic features of Alz‐ heimer's disease and, therefore, those biomarkers that can detect and quantify Aβ protein or tau protein accumulated in brain tissue are vitally important for the pathological diagnosis of the disease and its precursors [49]. Aβ deposition markers include both measurements of the level of Aβ42 in cerebrospinal fluid (CSF) as evidence of deposition via positron emission tomography (PET) imaging by using a variety of specific binding agents [50]. Tau protein

Among neuronal injury markers, there is a series of structural and functional measures,

*Other non-validated measures*: fMRI activation studies, resting BOLD functional connectivity, MRI perfusion, MR

*Abbreviations*: CSF, cerebrospinal fluid; PET, positron emission tomography; FDG, fluorodeoxyglucose; SPECT, single photon emission tomography; fMRI, functional magnetic resonance imaging; BOLD, blood oxygen level-dependent;

Among the potential uses of biomarkers, there is the identification of people in the preclinical stages of the disease or the reduction in the heterogeneity of the disease in clinical trials. However, not all biomarkers are valid as indirect assessment criteria, as they can be difficult to validate and require different levels of validation depending on their expected use. In this respect, although the presence of high levels of tau protein is clearly associated with the pathophysiological process of Alzheimer's disease, changes in tau and phosphorylated-tau (p-Tau) are not exclusive to Alzheimer's disease and can also reflect more general neuronal and synaptic damage. The same can be said of Aβ depositions, as they are not exclusive to Alzheimer's disease. This is one of the main limitations of these biomarkers [51]. No conclusive determination has been reached either concerning whether the quantitative measure of these biomarkers provides us with more information than the dichotomous assessment (presence/ absence; positive/negative). Nevertheless, the combination of both types of measures points

accumulation markers can be obtained through measurements via CSF.

described in **Table 3**, together with Aβ biomarkers.

**Biomarkers for the diagnosis of cognitive impairment**

106 Molecular Mechanisms of the Aging Process and Rejuvenation

**Biomarkers of Aβ deposition**

**Biomarkers of neuronal injury** - CSF tau/phosphorylated-tau

spectroscopy, and diffusion tensor imaging.

**Table 3.** Biomarkers for clinical diagnosis.

MR, magnetic resonance. Adapted from Albert et al. [29].

After thoroughly analysing the background and revising the main research studies carried out at the epidemiological, neuropsychological, genetic levels, and with biomarkers, the group of experts of the National Institute on Aging and the Alzheimer's Association proposed a working framework (**Figure 3**) to provide specialists, and the scientific world in general, with a specific and common language to move towards the knowledge of the preclinical and clinical stages of Alzheimer's disease.

The objective of the group of experts is to contribute with some operational research criteria in order to help select future target groups at risk of developing Alzheimer's disease, by considering the presence of Aβ biomarkers (either in isolation or in combination with other neurodegeneration markers). We have to consider that, at the moment, the use of these new criteria is for research only and that an inappropriate use in other fields may lead to wrong results and misinterpretations, as so far these criteria are still under revision.

These new criteria are based on the assumption that Alzheimer's disease is characterized by a sequence of biological events that begin much before the clinical symptoms of the disease appear. Considering the genetic evidence, the hypothesis of Aβ accumulation in a first stage of amyloidosis, which would be the first moment when cognitive impairment can be measured, is gaining strength. This first stage occurs before any other symptomatic manifestation and, therefore, would allow for a first diagnosis, which would indicate that a still indefinable process has started. At the same time, the new criteria acknowledge that the preclinical stages of Alzheimer's disease represent a continuum, including those cases that will never go beyond stage 1 or 2. The presence of one or two biomarkers, once the MCI stage has been reached, would indicate that an individual is experiencing a neurodegenerative process that in further stages will be defined.

As the group of experts highlights in their conclusions [32], at the moment, these criteria are to be used only in research and have no diagnostic utility; the objective of the new criteria is to allow researchers to better characterize the biological sequence that triggers Alzheimer's disease from the first moments. Therefore, these criteria have to facilitate the standardization of data collection in new studies, whose results will modify the very same criteria.

**Figure 3.** New proposal for research criteria for MCI and Alzheimer's disease.

#### **6. Neuropsychological contributions to the early detection of cognitive impairment**

Research has highlighted, beyond the genetic base, a series of factors that have a crucial significance in the development and course of cognitive impairment and Alzheimer's disease. There is great interest in knowing, for example, the association between two facts: cognitive impairment and metabolic and vascular alterations, such as cardiovascular diseases, high blood pressure, diabetes, or obesity, on the one hand, and the establishment and course of the disease, on the other. Understanding these relationships and obtaining data via clinical studies can help us understand that the fact of reducing risk factors associated to these pathologies may be also useful to control Alzheimer's disease. Moreover, a healthy balanced diet, physical exercise, social commitment, and mentally challenging activities can help people to keep healthy as they get older [53].

With regard to environmental factors, included in the diagram of **Figure 2**, different studies show that a higher educational level, complex working activities or a socially integrated lifestyle are factors that can help to postpone the onset of clinical cognitive impairment [54, 55]. It has also been shown that physical exercise and cardiovascular activity have long-term benefits on cognition [56]. Actually, physical activity has been proved to reduce the risk of developing dementia and to improve cognition [57].

On the other hand, vascular risk factors are associated, by definition, to vascular dementia. In contrast, the relationship between vascular risk factors and the development and progression of cognitive impairment associated with Alzheimer's disease is less clear. For more than three decades, Alzheimer's disease has been described as a primary neurodegenerative disorder with scarce, or none, vascular foundation [5, 58]. Nevertheless, in the last few years, knowledge about this relationship has increased a lot and many current studies relate vascular risk factors to the pathogenesis of mild cognitive impairment and Alzheimer's disease (see De la Torre for a thorough theoretical revision [59]).

Knowledge about the risk factors of Alzheimer's disease has expanded a lot, and at present, they include not only risk factors particular to the ageing and adulthood process but also risk factors particular to all life stages. For example, perinatal conditions, brain development, growth factors, socioeconomic conditions, or cognitive reserve are factors that have been shown to have an influence on the process of developing dementia [60, 61].

The identification of prodromal neuropsychological markers in cognitive impairment is based on the idea that neuronal loss starts much earlier than clinical symptoms. For this, it is essential to clinically detect individuals in the first stages of impairment, as starting treatment in these early moments would help to maximize the impact on maintaining cognitive functions and functional skills. Hence, the importance of having adapted tests to detect cognitive impairment at early stages, as an early diagnosis of cognitive impairment at prodromal stages is still a very important objective, considering the probability that this stage will be susceptible to treatments designed both to stop and to slow down the progression of the impairment.

**Figure 3.** New proposal for research criteria for MCI and Alzheimer's disease.

108 Molecular Mechanisms of the Aging Process and Rejuvenation

**impairment**

**6. Neuropsychological contributions to the early detection of cognitive**

Research has highlighted, beyond the genetic base, a series of factors that have a crucial significance in the development and course of cognitive impairment and Alzheimer's disease. There is great interest in knowing, for example, the association between two facts: cognitive impairment and metabolic and vascular alterations, such as cardiovascular diseases, high blood pressure, diabetes, or obesity, on the one hand, and the establishment and course of the disease, on the other. Understanding these relationships and obtaining data via clinical studies can help us understand that the fact of reducing risk factors associated to these pathologies may be also useful to control Alzheimer's disease. Moreover, a healthy balanced diet, physical

In summary, literature suggests that the risk of starting a process of cognitive impairment linked to Alzheimer's disease is not determined only by a genetic component or by certain risk factors in adulthood, but by the result of a complex interaction between genetic and environ‐ mental factors throughout our entire life.

In the next decades, an increase in the prevalence of Alzheimer's disease in particular and cognitive impairment in general is expected. Advances in clinical research will make the management of this disease more sophisticated. In the near future, there will be new tests to identify both people at risk of developing Alzheimer's disease and those having early symp‐ toms of cognitive impairment. At the same time, there will be more medicines available and possibly the progression of the disease may be delayed for years. With the improvement in diagnosis and early detection of Alzheimer's disease, more people will be diagnosed at the early stages of the disease. The role of the family will be more and more crucial, as together with early diagnosis and pharmacological treatment there will be coordination of care and support functions both for patients and for carers of people with dementia. Many of these individuals will have the added value of still being there for their families, to look after children, for example, or being an important part in the ageing of the parents themselves. To improve care and support to these people, the public and private sectors have to work together to eliminate age-related barriers that reduce access to help and support services and to improve the comprehension of the unique needs of the people diagnosed with Alzheimer's disease and their environment.

#### **Author details**

Judit Subirana-Mirete

Address all correspondence to<EMAIL_ADDRESS>
Faculty of Psychology, Education and Sport Sciences Blanquerna, Ramon Llull University, Barcelona, Spain

#### **References**

Junqué C, Ruano A, editors. Rehabilitación neuropsicológica [Neuropsychological rehabilitation]. Barcelona: Elsevier, Masson; 2011. pp. 289–318.

with early diagnosis and pharmacological treatment there will be coordination of care and support functions both for patients and for carers of people with dementia. Many of these individuals will have the added value of still being there for their families, to look after children, for example, or being an important part in the ageing of the parents themselves. To improve care and support to these people, the public and private sectors have to work together to eliminate age-related barriers that reduce access to help and support services and to improve the comprehension of the unique needs of the people diagnosed with Alzheimer's disease and

Faculty of Psychology, Education and Sport Sciences Blanquerna, Ramon Llull University,

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Address all correspondence to<EMAIL_ADDRESS>
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## *Edited by Naofumi Shiomi*

Numerous studies had been performed to elucidate the mechanisms of aging and to achieve rejuvenation, with some success reported in recent years. However, at present, the findings from those studies are not sufficient to resolve the issue of aging. This book presents an overview of recent topics on cellular aging and rejuvenation. In the early chapters, the molecular mechanisms of aging via the activities of clock and ion channel proteins, in addition to overall aspects, are discussed. In the latter part, the aging of the skin, immune system, and brain is discussed. This book will prove useful for those studying or developing new drugs to counter the aging process and will encourage the development of novel ideas for rejuvenation.

Photo by ktsimage / iStock

Molecular Mechanisms of the Aging Process and Rejuvenation

Molecular Mechanisms

of the Aging Process and

Rejuvenation

*Edited by Naofumi Shiomi*