Here, we set to evaluate whether mice with short telomeres in the brain owing to telomerase deficiency could be used as a bona fide neurodegeneration model. To this end, we characterized the molecular consequences of the presence of short telomeres in the neurogenic areas of the brain as well as the potential cognitive defects associated to short telomeres in these mice. We also explored whether similar signs of neurodegeneration can be found in wild-type mice associated to physiological mouse aging. Finally, we demonstrate that telomerase activation using AAV9- Tert gene therapy can ameliorate the brain phenotypes in these mice .

In spite of the fact that the brain is a low-proliferative tissue, there are regenerative areas within the brain such as the hippocampus, the subventricular zone (SVZ), and the olfactory bulb. Interestingly, several studies suggest the presence of short telomeres in patients with advanced Alzheimer’s disease [ 27 – 32 ]. In the case of Parkinson’s disease, extensive research has not been performed. A few studies have found a correlation between short telomere length and Parkinson’s disease [ 33 , 34 ], while other studies found no correlation [ 35 , 36 ]. These studies were performed with peripheral blood leukocytes, and telomere length was not measured in the brain cells implicated in Parkinson’s disease. In mice, we have previously shown that telomere attrition in the context of the Terc -deficient mouse model [ 19 ] impairs in vitro proliferation of adult neural stem cells from the SVZ [ 37 ], and that telomere shortening in mice disrupts neuronal differentiation and neurogenesis [ 38 ]. These findings pose the interesting hypothesis that telomerase reactivation in the brain may have significant therapeutic effects. Indeed, both in our AAV9- Tert mediated telomerase reactivation model [ 23 ] and in a genetic Tert reactivation mouse model [ 39 ], there were beneficial effects associated to telomerase reactivation in the brain. In particular, telomerase re-expression in a telomerase-deficient mouse model with short telomeres, resulted in a larger brain weight, a thicker myelin sheath, better performance in an innate avoidance test as a measure of the health of the olfactory bulb, and increased molecular markers of Ki67, Sox2, doublecortin, and Olig2 [ 39 ]. Similarly, telomerase re-expression in the brain using AAV9 gene therapy in adult wild-type mice was sufficient to improve cognitive function [ 23 ].

In support of critically short telomeres being a determinant of aging and longevity, we previously showed that increased TERT expression in the context of cancer resistant transgenic mice was sufficient to delay aging and extend mouse longevity by 40% [ 22 ]. We further demonstrated that telomerase reactivation in adult tissues by using adeno-associated viruses (AAV9- Tert ) was able to significantly delay age-related diseases and increase longevity in wild-type mice [ 23 ]. In particular, AAV9- Tert treatment resulted in reduced age-related osteoporosis, reduced glucose intolerance, increased neuromuscular coordination, enhanced memory in an object recognition test, improved mitochondrial fitness, and delayed cancer, thus demonstrating that telomere shortening is causative of aging and is at the origin of a wide range of age-associated diseases, including cognitive decline [ 23 ]. More recently, we have also shown that AAV9-mediated telomerase activation has therapeutic effects in pre-clinical mouse models for diseases associated with short telomeres such as aplastic anemia [ 24 ], myocardial infarction [ 25 ], and pulmonary fibrosis [ 26 ].

Telomeres are heterochromatic protective structures at the ends of chromosomes that consist of TTAGGG repeats bound by a six-protein complex known as shelterin [ 4 , 5 ]. A minimum length of telomeric repeats is necessary for shelterin binding and protection [ 4 , 5 ]. Telomerase is a reverse transcriptase which can elongate telomeres de novo by the addition of telomeric repeats onto chromosome ends [ 6 ], in this manner compensating progressive telomere attrition as a consequence of cell division. Telomerase is composed of two essential subunits, the telomerase reverse transcriptase (TERT) and the telomerase RNA component (Terc), which is used as a template for the synthesis of telomeric repeats [ 6 ]. Adult tissues, including the stem cell compartments, do not have sufficient telomerase activity to compensate for telomere shortening associated with tissue regeneration and cell division [ 7 – 9 ]. When telomeres reach a critically short length, this triggers activation of a persistent DNA damage response at telomeres and the subsequent induction of cellular senescence or apoptosis [ 5 , 10 ]. Short/dysfunctional telomeres are considered one of the primary hallmarks of aging both in mice and humans, as they lead to other hallmarks of aging, such as genomic instability, cellular senescence, and loss of the regenerative capacity of tissues [ 11 ]. In particular, critically short telomeres in the stem cell compartments lead to impaired tissue renewal and homeostasis [ 12 – 14 ]. Interestingly, the rate of telomere shortening throughout lifespan is influenced by both genetic factors (i.e. mutations in genes necessary for telomere maintenance) and environmental factors (i.e. cigarette smoke has a negative effect) [ 15 , 16 ]. Interestingly, there are a number of diseases associated to mutations in telomerase and other telomere maintenance genes known as “telomere syndromes”, which include dyskeratosis congenita, aplastic anemia and pulmonary fibrosis, among others (for a review see [ 17 ]). These syndromes are characterized by the presence of extremely short telomeres, which prematurely impair the regenerative capacity of tissues, affecting both high and low proliferative tissues [ 17 , 18 ]. Prior to the discovery of human “telomere syndromes”, similar findings were made by studying mice genetically modified to lack the telomerase RNA component ( Terc -/- ) [ 19 ]. Terc -deficient mice have shorter telomeres with increasing mouse generations and this results in a progressive decrease of both median and maximum longevity [ 20 , 21 ]. These mice show pre-mature appearance of different pathologies affecting both proliferative and non-proliferative tissues, which are accompanied by an impaired regenerative capacity (reviewed in [ 10 , 13 ]).

Parkinson’s, are associated with aging, and their pre-valence is increasing as there are more individuals that reach older ages [ 1 – 3 ]. To date, there are no curative treatments for any of these diseases owing to the fact that their molecular origins are still poorly understood. Instead, palliative treatments are directed to alleviate downstream events, such as beta-amyloid deposition in the case of Alzheimer’s or lack of dopamine generation in the case of Parkinson’s due to the loss of dopaminergic neurons.

Results

Histological and molecular defects associated to shorter telomeres in the brain of telomerase-deficient mice First, we set to characterize brain phenotypes in the telomerase-deficient Tert-/- mouse model used in this study [40–43]. We first weighed the brains of age-matched wild-type and third generation (G3) Tert-/- mice, which have shorter telomeres owing to telomerase deficiency for three generations. We observed that the brains of G3 Tert-/- mice showed a tendency to be smaller than those of age-matched wild-type controls (Figure 1A), in agreement with a previous report [39]. A reduced brain size may be indicative of neuro-degeneration as this phenotype is also observed in patients with advanced Alzheimer’s disease [44]. In this regard, we quantified the size of the hippocampus and dentate gyrus (DG) of the different mouse cohorts (Figure 1B). We found that G3 Tert-/- mice also show smaller hippocampus and dentate gyrus regions than wild-type mice (Figure 1B). Figure 1. Mice deficient for telomerase have smaller brains, shorter telomeres, more proliferation, more DNA damage, and less neurogenesis. (A) Brain weight and representative images of young and old wild-type and G3 Tert-/- mice. (B) Area of hippocampus and dentate gyrus in untreated mice quantified from representative images of brain sections stained with hematoxylin and eosin. (C) Q-FISH for telomere spot fluorescence measured in the hippocampus, (D) the dentate gyrus specifically, (E) the subventricular zone, and (F) the neocortex. The mean telomere spot fluorescence is shown. The percentage of short telomeres is also shown with “short” being defined as a fluorescence intensity less than the 15th percentile of the fluorescence intensity values of a control sample. Cartoon diagrams label the different regions of the brain. In part (C), A scan of a coronal brain cross-section without fluorescence is shown with the hippocampus region highlighted in yellow. Representative images show the telomere spots labeled with Cy3-Tel probe (in red), and nuclei stained with DAPI (blue).

We next set to characterize the molecular events associated to telomerase deficiency in the brain of G3 Tert-/- mice. We first determined telomere length in several brain regions of age-matched wild-type and G3 Tert-/- mice, including the hippocampus since this structure is critical for learning, memory, memory for episodic events, and neurogenesis [45,46], as well as the DG area within the hippocampus since this region is involved in neurogenesis [47–51] (see scheme in Figure 1C). In addition, we also studied telomere length in the subventricular zone which is important for neurogenesis in the adult brain [52], and the neocortex which is important for higher-order brain functions such as sensory perception, cognition, generation of motor commands, spatial reasoning, and language [53]. To this end, we performed quantitative telomere FISH (Q-FISH) directly on coronal paraffin brain sections from young wild-type (8-10-weeks old), old wild-type (71-weeks old), young G3 Tert-/- (14-weeks old), and old G3 Tert-/- mice (69-72-weeks old). We found that telomeres were significantly shorter in the hippocampus, dentate gyrus, subventricular zone, and neocortex of G3 Tert-/- mice compared to the same regions in wild-type mice (Figure 1C-F). Accordingly, we also found that the percentage of short telomeres in G3 Tert-/- in these brain regions was higher (Figure 1C-F). Short telomeres were defined as telomeres with a fluorescence intensity less than the 15th percentile of the intensity values of a control. Next, we determined whether shorter telomeres in these brain regions of Tert-/- mice were associated to reduced proliferation and increased DNA damage [54]. To this end, we used immunohistochemistry to determine the number of Ki67-positive cells as a marker of cycling cells and the number of cells positive for γH2AX as a marker for DNA damage. In the case of wild-type mice, we observed significantly fewer Ki67-positive cells in older mice (71-weeks old) compared to younger ages (8-weeks old) in the hippocampus, the dentate gyrus, subventricular zone (SVZ) of the lateral ventricle anterior to the hippocampus level, and the neocortex (Figure 2A-C). Interestingly, young G3 Tert-/- mice (14-weeks old) also showed lower numbers of Ki67-positive cells than age-matched wild-type controls in these brain regions, and this was further reduced in the old G3 Tert-/- mice (Figure 2A-C). Regarding DNA damage, we found increased numbers of γH2AX-positive cells in the hippocampus, dentate gyrus, and SVZ of old wild-type mice (71-weeks old) compared to young mice (8-weeks old) (Figure 2D,E). Young G3 Tert-/- mice also showed significantly lower numbers of γH2AX-positive cells in the hippocampus, dentate gyrus, and subventricular zone compared to older G3 Tert-/- mice (Figure 2D,E). In the neocortex, more γH2AX associated DNA damage was only observed in the old G3 Tert-/- mice (Figure 2F). These results indicate that physiological aging in wild-type mice, as well as accelerated telomere shortening as a consequence of telomerase-deficiency in the Tert-/- mice, lead to decreased proliferation and increased DNA damage in the hippocampus, dentate gyrus, SVZ, and neocortex. Figure 2. Immunohistochemistry of Ki67 and γH2AX in the brain. (A-F) The quantification and representative images of the immunohistochemistry for positive cells per field of view for (A-C) Ki67, and (D-F) γH2AX in brain regions such as the hippocampus, dentate gyrus, subventricular zone (SVZ) of the lateral ventricle anterior to the hippocampus level, and the neocortex. The data is shown for young and old wild-type and G3 Tert-/- mice. Data represent the mean ±SE of analyzed mice within each group. For the histopathology results, the number of mice analyzed per group is indicated, as well as the number of fields of view, and the number of positive cells. The t-test was used for statistical analysis. *p<0.05; **p<0.01; ***p<0.001.

Next, we set to address the impact of shorter telomeres on neurogenesis, inflammation, and formation of tau protein aggregations. In this regard, neurogenesis has been suggested to act as a brain repair mechanism which could mitigate the effects of neurodegeneration that occurs with Alzheimer’s disease and possibly aging [54,55]. Doublecortin is expressed in developing neurons and it is considered a bona fide marker of neurogenesis [39,56]. To this end, we set to study the expression of doublecortin in various brain regions of both wild-type and G3 Tert-/- mice. We found that the number of cells and G3 Tert-/- mice. We found that the number of cells expressing doublecortin was significantly decreased in expressing doublecortin was significantly decreased in old wild-type mice (71-weeks old) compared to young (8-weeks old) wild-type mice in the hippocampus and the SVZ, and showed the same tendency in the dentate gyrus and the neocortex (Figure 3A-C). Young G3 Tert-/- mice also showed lower numbers of cells ex-pressing doublecortin in the hippocampus, dentate gyrus, subventricular zone, and neocortex compared to age-matched wild-type controls, and this was further aggravated with increasing age (Figure 3A-C). The same trends were observed for doublecortin expression in the parietotemporal region of the cerebral cortex (Supplementary Figure S1A), and the occipital region of the cerebral cortex (Supplementary Figure S1B). Neurogenic niches were also identified in hematoxylin and eosin stains in the parietal subventricular zone area, and the young wild-type mice had higher levels of neurogenic niches than old wild-type mice (Supplementary Figure S1C). In agreement with this finding, young G3 Tert-/- mice had more neurogenic niches than old G3 Tert-/- mice, but less than young wild-type mice (Supplementary Figure S1C). These findings indicate that shorter telomeres associated with aging and to telomerase-deficiency correlate with impaired neurogenesis in different regions of the brain. Figure 3. Immunohistochemistry of GFAP and Tau in the brain. (A-F) The quantification and representative images of the immunohistochemistry for positive cells per field of view for (A-C) doublecortin, (D-E) glial fibrillary acidic protein (GFAP), and (F) p-Tau(Ser396) in brain regions such as the hippocampus, dentate gyrus, and the neocortex. The data is shown for young and old wild-type and G3 Tert-/- mice. Data represent the mean ±SE of analyzed mice within each group. For the histopathology results, the number of mice analyzed per group is indicated, as well as the number of fields of view, and the number of positive cells. The t-test was used for statistical analysis. *p<0.05; **p<0.01; ***p<0.001.

A sign of brain aging is neuroinflammation [57]. In particular, the expression of glial fibrillary acidic protein (GFAP) by astrocytes increases with aging as astrogliosis and neuroinflammation occurs, and such an increase is also observed in mouse models with in-creased neuroinflammation [58–60]. The increase in GFAP expression accompanies increased expression of inflammatory cytokines, accumulation of proteotoxic aggregates, and senescence [58]. To address, whether physiological aging and/or short telomeres in the context of telomerase deficiency lead to increased inflammation in the brain, we measured the number of immune astrocyte cells with strong expression of the GFAP marker [61]. We observed that young wild-type mice have low levels of GFAP in the hippocampus and dentate gyrus, whereas old wild-type, young G3 Tert-/-, and old G3 Tert-/- mice have more GFAP-positive astrocytes in these areas (Figure 3D). We did not find significantly more GFAP in the neocortex of G3 Tert-/- mice compared to wild-type, but did find an increase of GFAP with age in both generations (Figure 3E). Another sign of brain aging is the accumulation of tau or abnormal phosphorylation of tau protein located throughout the brain in various cell types such as neurons, astrocytes, and oligodendrocytes, ultimately resulting in aggregates and neurofibrillary tangles [62–64] . Indeed, tau has been associated with Alzheimer’s because hyperphosphorylation of tau results in loss of its biological activity [65]. To address this in our mouse models, we measured protein aggregation by determining the number of cells positive for tau phosphorylated at serine 396 [66,67]. We observed more cells positive for phosphorylated tau in the hippocampus and dentate gyrus of old wild-type mice, and this effect was further increased in old G3 Tert-/- mice (Figure 3F). In summary, these findings indicate that short telomeres in the brain contribute to impair brain neurogenesis and to increase neuroinflammation and abnormal tau phosphorylation. Interestingly, similar phenotypes were observed associated to physiological aging in very old wild-type mice.

Telomerase-deficient mice are more susceptible to MPTP neurotoxin To determine whether mice deficient for telomerase were more susceptible to cellular damage, we per-formed an experiment with the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) neurotoxin. MPTP specifically damages dopaminergic neurons in the substantia nigra region of the brain [68]. Inside these neurons, MPTP is metabolized to MPP+ which then interferes with complex I of the electron transport chain in mitochondria, ultimately resulting in oxidative damage. The MPTP neurotoxin model is a common model used to study Parkinson’s disease [69]. In our experiment, 12-16-week old male wild-type and G3 Tert-/- mice were injected IP (intraperitoneally) with 0.5, 5, 10, or 20 mg/kg MPTP and sacrificed 7 days later (scheme in Supplementary Figure S2A). An additional injection was administered 2 hours later since multiple injections have been suggested by previous protocols [69]. A footprint test resulted in a trend showing that wild-type and G3 Tert-/- mice injected with the MPTP had a shorter hind paw stride length (Supplementary Figure S2B). In addition, we performed a tail suspension test to measure mobility. In particular, we hung the mice from the tail for a period of 5 min and measured their immobility time. Mice that are old, unhealthy, or more depressed, move less in the tail suspension test [70]. We found that wild-type mice exhibited a shorter immobility time than MPTP-treated wild-type and G3 Tert-/- mice (Supplementary Figure S2C). After 7 days, the mice were sacrificed, and the brain was preserved with formalin. In order to investigate the dopaminergic neurons, we performed an immuno-fluorescence experiment for the tyrosine hydroxylase marker. Tyrosine hydroxylase is important in dopaminergic neurons since it converts the amino acid L-tyrosine to L-DOPA which is a precursor for dopamine [71]. We found that 12-16-week old male mice injected with higher doses of MPTP had lower levels of tyrosine hydroxylase (Figure S2D). Interestingly, saline-treated control G3 Tert-/- mice showed less tyrosine hydroxylase than wild-type mice (Figure S2D), and G3 Tert-/- mice treated with a low dose of MPTP showed less styrosine hydroxylase than similarly treated wild-type mice (Figure S2D). Additionally, old wild-type and old G3 Tert-/- mice had lower levels of tyrosine hydroxylase than young mice (Supplementary Figure S2E). Together these results suggest that G3 Tert-/- mice are more susceptible to damage to dopaminergic neurons in the substantia nigra due to MPTP neurotoxin, and this damage results in decreased mobility.

Behavioral defects associated to shorter telomeres in the brain of telomerase-deficient mice We previously showed that old wild-type mice perform more poorly than young mice in tests such as the object recognition test, rotarod test, and tightrope test [23], suggesting a negative impact of telomere shortening associated with aging in these tests. Thus, we here set to address whether telomerase deficient mice performed more poorly than age-matched wild-type controls in different behavioral and cognitive tests. The tightrope test measures the ability of mice to balance on a tightrope, and the rotarod test measures neuromuscular coordination while running on a rotating rod. In addition, we performed a tail suspension test to measure mobility as described previously in this text. We found that late generation G1, G2, G3, and G4 Tert-/- mice performed worse than age-matched wild-type mice in the tightrope test and rotarod test at older ages (Supplementary Figure S3A,B). Furthermore, we found that older G2 Tert-/- mice are immobile in the tail suspension test for longer periods of time than age-matched wild-type mice and this was further aggravated in the G3 and G4 Tert-/- mice (Supplementary Figure S3C), suggesting a negative impact of progressively shorter telomeres in this test. Thus, telomerase deficiency and shorter telomeres lead to significantly impaired performance in several tests that measure neuromuscular coordination and balance. To further assess a potential impact of telomerase deficiency on cognitive function, we subjected the different mouse cohorts to an object recognition test. We found that G4 Tert-/- mice spent less time investigating a novel object in their environment than age-matched wild-type mice (Supplementary Figure S3D), which is an indication of poor memory in G4 Tert-/- mice with shorter telomeres [72]. Together, these findings indicate that mice with shorter telomeres show noticeable behavioral phenotypes consisting of a poorer performance in behavioral and cognitive tests. Finally, we set to analyze the brain metabolic activity in the different mouse cohorts as another indication of brain defects in these mice. To this end, we compared differences in metabolic activity in the brains of wild-type and G3 Tert-/- mice by using positron emission tomography (PET) to detect fluorodeoxyglucose (FDG) in the brain after injection. Brain glucose metabolism has been found to decrease in human patients with Alzheimer’s disease and during senile dementia [73–75]. We found that G3 Tert-/- mice had a lower standard glucose uptake value (SUV) in the brain compared to wild-type brains, which is an indication of lower metabolic activity (Supplementary Figure S3E). The weights of the mice were taken into account when calculating the SUV. Thus, telomerase deficiency and shorter telomeres resulted in decreased glucose metabolism in the brain, which is associated with poorer cognitive performance and increased neurodegeneration. Together, these findings show that Tert-/- mice with short telomeres exhibit more neurodegeneration than wild-type mice, suggesting an impact of telomere length on brain aging in mice.

AAV9-mediated telomerase transduction of mouse brains Next, we set to study whether expression of telomerase in these mouse models could ameliorate the signs of neurodegeneration described above. Previous reports have shown that adeno associated viruses of the serotype 9 (AAV9) are able to cross the blood brain barrier and transduce a significant percentage of cells [76–84]. To verify whether in our experimental setting an IV (intravenous) injection of the AAV9 vector could cause effective expression of the Tert transgene in the brain, we injected 8-week old mice with the AAV9-CMV-Tert, AAV9-CMV-eGFP, or AAV9-CAG-eGFP vector, and the mice were sacrificed after 2 weeks of treatment to study expression of the eGFP or Tert transgenes (scheme in Figure 4A). The “CMV” abbreviation corresponds to the cytomegalovirus promoter, and the “CAG” abbreviation corresponds to the high-expression synthetic CAG promoter [85]. We first determined whether AAV9 carrying either eGFP or Tert genes caused gene expression in the mouse brain upon intravenous (IV) injection in the tail with a dose of 2E12 viral genomes (vg) per mouse (see Methods). To this end, 8-week old mice were injected with AAV9 virus particles containing either Tert or eGFP (Figure 4A) and under either the CMV promoter [23–25], or the CAG promoter. Mice treated with the different viral vectors were sacrificed 2 weeks after tail injection and several different analyses were performed. We performed quantitative qPCR to detect Tert and eGFP mRNA expression in the brain (Figure 4B). We found expression of the eGFP gene in the brains of AAV9-eGFP treated mice but not in brains from the AAV9-Tert treated mice (Figure 4B). In turn, the Tert mRNA was only detected in the brain of AAV9-Tert treated mice but not in those treated with AAV9-eGFP (Figure 4B). We also measured eGFP fluorescence in the whole mouse, including the brain, using an IVIS instrument (Figure 4C). AAV9-eGFP treated mice displayed detectable eGFP fluorescence in the body as well as in the brain, whereas untreated wild-type control mice only displayed background levels of fluorescence (Figure 4C). Thus, these results confirmed that AAV9 viruses carrying either the eGFP or Tert genes were able to cross the blood-brain barrier and infect cells in the brain. Figure 4. Experiment scheme and confirmation of virus transduction in the brain. (A) Scheme of the gene therapy experiment. Wild-type mice (8-week old) were injected IV with AAV9-CMV-Tert, AAV9-CMV-eGFP, or AAV9-CAG-eGFP, and sacrificed 2 weeks after injection. (B) Level of eGFP and Tert mRNA relative to actin in the brain as measured by qPCR 2 weeks post IV tail injection with 2E12 vg of AAV9-CMV-Tert, AAV9-CMV-eGFP, AAV9-CAG-eGFP, or no virus. (C) Quantification and representative images of fluorescence as measured by an IVIS instrument in the body and brain of wild-type mice and mice injected IV in the tail with AAV9-CMV-eGFP. (D) Quantification and representative images of eGFP positive cells in the brain as measured by immunohistochemistry in mice injected in the tail with 2E12 vg AAV9-CAG-eGFP or no virus. The percentage of eGFP positive cells was calculated from the whole coronal brain cross-section. The representative images show multiple regions throughout the brain as labelled. (E) Quantification and representative images of eGFP positive cells in the brain as measured by immunofluorescence in mice injected IV in the tail with 2E12 vg AAV9-CMV-eGFP, AAV9-CAG-eGFP, or no virus. The percentage of eGFP positive cells was calculated from the whole coronal brain cross-section. The representative images show multiple regions throughout the brain as labelled with DAPI stained nuclei in blue, eGFP in green, and Iba1 for microglia in red. (F) Scheme of experiment with injection of AAV9-Tert into young mice. The mice (wild-type, G3 Tert-/-, and G4 Tert-/- mice) were treated at a young age (27-30 weeks) by the IV tail injection of 2E12 vg of AAV9-Tert or AAV9-null virus. (G) The mRNA level of Tert in the brain at the humane endpoint. The mRNA level was measured by qPCR relative to GAPDH. The ages of the mice as well as the number of weeks of treatment for each group are indicated above the graph. Data represent the mean ±SE of analyzed mice within each group. The number of mice analyzed per group is indicated. The t-test was used for statistical analysis. *p<0.05; **p<0.01.

Note that the AAV9-CMV-eGFP virus should infect the same number of cells as the AAV9-CAG-eGFP since the viral capsid is the same. However, the CMV promoter results in lower levels of expression, and therefore we are unable to detect the AAV9-CMV-eGFP infected cells by immunohistochemistry or immunofluorescence. Therefore, the AAV9-CAG-eGFP vector was utilized to locate infected cells and determine the number of cells that were infected. We could not use an AAV9-CAG-Tert vector because the genetic material is too large to efficiently pack into the viral vector. Next, in order to visualize eGFP expression in different brain areas by immunohistochemistry and immuno-fluorescence, we used the AAV9-CAG-eGFP vector, previously shown to have a very high expression level in the brain ([86,87]; see also Figure 4C). Upon tail injection, upon tail injection of AAV9-CAG-eGFP vectors, we used immunohistochemistry to detect eGFP positive cells throughout the entire brain, including the dentate gyrus, CA1 of the hippocampus, CA3 of the hippocampus, subventricular zone (SVZ) of the lateral ventricle, the neocortex, and the thalamus, which showed the highest eGFP expression (Figure 4D). Transduction of brain cells was also confirmed by immunofluorescence using an antibody against GFP, including all the brain areas studied (Figure 4E). We observed that the transduction efficiency of all cells in a whole brain cross-section from a position in the brain at the hippocampus level was approximately 2.5% as detected by immunohistochemistry (Figure 4D) and approximately 0.4% as detected by immunofluorescence (Figure 4E). This provides a range for the transduction efficiency which is in approximately the same range reported by other studies using different promoters [77,88]. We also addressed whether AAV9 transduced microglia which express the marker Iba1. However, we did not detect significant eGFP expression in the Iba1-positive microglia cells, also in agreement with previous reports [77,89]. Next, to address whether telomerase over-expression in the adult brain of mice could ameliorate signs of brain damage and neurodegeneration in mice, age-matched (27-30-weeks of age) wild-type, G3 Tert-/-, and G4 Tert-/- mice were treated with an IV tail injection of 2E12 vg of AAV9-Tert or AAV9-null vectors (scheme in Figure 4F). The mice were then followed throughout their lifespan until they reached the humane endpoint at which time we performed a number of molecular determinations. First, and in agreement with transduction of mouse brain, qPCR analysis demonstrated increased Tert mRNA expression in the brains of mice transduced with the AAV9-Tert vectors compared to those transduced with the null-vectors at the time of death (Figure 4G). Thus, increased Tert expression was maintained throughout the lifespan of the mice even until the humane endpoint. A graph of the body weights of the mice is presented in Supplementary Figure S4.

AAV9-Tert gene therapy results in less signs of molecular aging in the brain in mice First, we assessed the presence of DNA damage at the humane endpoint using the γH2AX marker. We confirmed more cells with DNA damage with increasing Tert-/- generations compared to wild-type mice in the hippocampus, the dentate gyrus, and the neocortex of mice treated with AAV9-null vectors (Figure 5A,B). Interestingly, we observed significantly lower numbers of cells with DNA damage for wild-type, G3 Tert-/-, and G4 Tert-/- mice groups treated with AAV9-Tert in the hippocampus, dentate gyrus, and neocortex compared to the controls treated with the AAV9-null vectors (Figure 5A,B). We also observed reduced levels of the senescence and DNA damage marker Trp53 mRNA in the AAV9-Tert treated G4 Tert-/- mice compared to the AAV9-null treated controls (Figure 5C), again suggesting less DNA damage in the telomerase-treated brains. We also addressed whether telomerase treatment had any impact on the regenerative neurons of the brain by quantifying cells positive for the neurogenesis marker doublecortin (Figure 6A,B). As expected, in the AAV9-null treated controls, we found significantly lower numbers of cells positive for doublecortin in the hippocampus and dentate gyrus, and of G4 Tert-/- mice compared to the wild-type mice (Figure 6A,B). Interestingly, AAV9-Tert treatment resulted in significantly increased numbers of doublecortin-positive cells in the hippocampus, the dentate gyrus, and the neocortex in G4 Tert-/- mice compared to those treated with AAV9-null vectors (Figure 6A,B). Differences were not observed between the wild-type groups, which died at a very old age (Figure 6A,B). Figure 5. Treatment with AAV9-Tert results in less DNA damage in the brain. (A-B) Quantification and representative images of the histopathology for γH2AX in the (A) hippocampus, (A) dentate gyrus, and (B) neocortex for the cohort of mice (wild-type, G3 Tert-/-, and G4 Tert-/- mice) injected with 2E12 vg of AAV9-Tert or AAV9-null. (C) The level of Trp53 mRNA in the brain as measured by qPCR relative to GAPDH at the humane endpoint. Data represent the mean ±SE of analyzed mice within each group. The number of mice analyzed per group is indicated. The t-test was used for statistical analysis. *p<0.05; **p<0.01; ***p<0.001.

Figure 6. Treatment with AAV9-Tert results in more neurogenesis and less inflammation in the brain. (A-B) Quantification and representative images of the histopathology for doublecortin in the hippocampus (A), dentate gyrus (A), and neocortex (B). (C) Immunofluorescence of tyrosine hydroxylase in the substantia nigra. (D-E) Quantification and representative images of the histopathology for glial fibrillary acidic protein (GFAP) in the hippocampus (D), dentate gyrus (D), and (E) neocortex. Data represent the mean ±SE of analyzed mice within each group. The number of mice analyzed per group is indicated. The t-test was used for statistical analysis. *p<0.05; **p<0.01; ***p<0.001.

We next evaluated dopaminergic neurons responsible for motor control and important in Parkinson’s disease by quantifying the tyrosine hydroxylase marker (Figure 6C). We found that G3 Tert-/- mice treated with AAV9-Tert had a higher intensity of tyrosine hydroxylase fluorescence than those treated with the AAV9-null vector (Figure 6C). Finally, we determined the number of GFAP-positive cells which is indicative of neuroinflammation. We found significantly lower levels of cells positive for GFAP in the hippocampus, dentate gyrus, and neocortex in G3 Tert-/- mice treated with the AAV9-Tert virus compared to the AAV9-null treated controls (Figure 6D,E). Thus, the AAV9-Tert therapy shows a trend towards reducing the level of inflammation in the brain, which reaches statistical significance between some of the groups. In summary, the fact that we observed fewer cells with DNA damage, higher levels of neurogenesis, less inflammation, and more tyrosine hydroxylase in dopaminergic neurons indicates that the AAV9-Tert gene therapy had an impact on these molecular markers of aging in the brain.