Gene therapy for Alzheimer's disease targeting CD33 reduces amyloid beta accumulation and neuroinflammation.

Neuroinflammation is a key contributor to the pathology of Alzheimer's disease (AD). CD33 (Siglec-3) is a transmembrane sialic acid-binding receptor on the surface of microglial cells. CD33 is upregulated on microglial cells from post-mortem AD patient brains, and high levels of CD33 inhibit uptake and clearance of amyloid beta (Aß) in microglial cell cultures. Furthermore, knockout of CD33 reduces amyloid plaque burden in mouse models of AD. Here, we tested whether a gene therapy strategy to reduce CD33 on microglia in AD could decrease Aß plaque load. Intracerebroventricular injection of an adeno-associated virus (AAV) vector-based system encoding an artificial microRNA targeting CD33 (miRCD33) into APP/PS1 mice reduced CD33 mRNA and TBS-soluble Aß40 and Aß42 levels in brain extracts. Treatment of APP/PS1 mice with miRCD33 vector at an early age (2 months) was more effective at reducing Aß plaque burden than intervening at later times (8 months). Furthermore, early intervention downregulated several microglial receptor transcripts (e.g. CD11c, CD47 and CD36) and pro-inflammatory activation genes (e.g. Tlr4 and Il1b). Marked reductions in the chemokine Ccl2 and the pro-inflammatory cytokine Tnfα were observed at the protein level in the brain of APP/PS1 mice treated with miRCD33 vector. Overall, our data indicate that CD33 is a viable target for AAV-based knockdown strategies to reduce AD pathology. One Sentence Summary: A gene therapy approach for Alzheimer's disease using adeno-associated virus vector-based knockdown of CD33 reduced amyloid beta accumulation and neuroinflammation.

Uterotrophic assay of percutaneous lavender oil in immature female rats.

The estrogenic potential of lavender oil was evaluated in a percutaneous uterotrophic bioassay in immature female rats. Four groups of 10 immature female rats each were randomly selected on postpartum day (PPD) 16. During the 3-day treatment period (PPDs 19-21), the immature rats were separated from the dams, caged in groups of 5 in a litter box for 6 hours, and administered the vehicle control article (corn oil) or lavender oil at 20 or 100 mg/kg per day. All dosages were administered as a 5 mL/kg volume in a Hilltop Chamber (25 mm diameter; absorbent material removed) placed on the shaved back of each immature rat, and secured with micropore tape and Vetrap. A positive control group was gavaged twice daily with 2.5 µg/kg per day of 17α-ethinyl estradiol. Daily observations included viability, clinical signs, body weights, and body weight gains. All rats were euthanized 24 hours after the third and final treatment, the uteri and ovaries were removed, and the paired ovaries and wet and blotted uterine weights were recorded. No unscheduled deaths occurred. No skin reactions were observed. Both dosages of lavender oil significantly reduced body weight gains after the third day of treatment, but terminal body weights and mean absolute and relative uterine weights did not differ significantly from vehicle control values. Positive controls showed significant increases in body weight and increased mean absolute and relative uterine weights as expected. Based on these data, lavender oil, at dosages of 20 or 100 mg/kg, was not active in the rat uterotrophic assay and gave no evidence of estrogenic activity.

Evaluation of the developmental toxicity of 4-tert-butylcyclohexyl acetate in Sprague-Dawley rats.

The fragrance ingredient 4-tert-butylcyclohexyl acetate (4-tBCHA) was evaluated for potential developmental toxicity in pregnant rats at oral dosages of 0, 40, 160, or 640 mg/kg per d in corn oil on gestational days 7 to 20. Increased salivation was observed at 160 and 640 mg/kg per d. The 640 mg/kg per d dosage was associated with the presence of a red perioral substance, ungroomed, sparse hair coat on the limbs, localized alopecia, reduced feed and body weight gains, or body weight losses, and mortality. Fetal body weights also were reduced at 640 mg/kg per d. This effect was associated with transient delays in fetal development, including significant increases in fetal incidences of moderate enlargement of the renal pelvis and reversible delays in ossification of the caudal vertebrae, fore and hind limb phalanges, and hind limb metatarsals. Maternal and developmental no observable adverse effect levels (NOAELs) were 160 mg/kg per d. It was concluded that 4-tBCHA is not a developmental toxicant in rats.

Cromolyn sodium delays disease onset and is neuroprotective in the SOD1G93A Mouse Model of amyotrophic lateral sclerosis.

Accumulating evidence suggests that neuroinflammatory processes are implicated in the initiation and progression of amyotrophic lateral sclerosis (ALS). Previous reports have demonstrated an increase in microgliosis and astrogliosis in the lumbar spinal cord of SOD1G93A transgenic mice before the onset of symptoms, a neuroinflammatory response which correlated with disease progression. Importantly, early stage homeostatic microglia enhanced motor neuron survival, while pro-inflammatory microglia were toxic to motor neurons in the SOD1G93A mice. Recent studies from our group have demonstrated that cromolyn sodium, an FDA approved compound, exerts neuroprotective effects in mouse models of Alzheimer's disease by altering microglial cell activation. Here, we tested the neuroprotective and anti-inflammatory effects of cromolyn sodium in the SOD1G93A mouse model of ALS. Our results indicate that cromolyn sodium treatment significantly delayed the onset of neurological symptoms, and improved deficits in PaGE performance in both male and female mice, however, there was only an effect on survival in female mice. Furthermore, there was a significant increase in motor neuron survival in the lumbar spinal cord as well as a significant decrease in the denervation of the neuromuscular junction of the tibialis anterior muscle in cromolyn treated transgenic SOD1G93A mice. Lastly, cromolyn treatment decreased the expression of pro-inflammatory cytokines/chemokines in the lumbar spinal cord and plasma and decreased mast cell degranulation in the tibialis anterior muscle of transgenic SOD1G93A mice. Together, these findings suggest that cromolyn sodium provides neuroprotection in the SOD1G93A mice by decreasing the inflammatory response.

TREM2 Acts Downstream of CD33 in Modulating Microglial Pathology in Alzheimer's Disease.

The microglial receptors CD33 and TREM2 have been associated with risk for Alzheimer's disease (AD). Here, we investigated crosstalk between CD33 and TREM2. We showed that knockout of CD33 attenuated amyloid beta (Aß) pathology and improved cognition in 5xFAD mice, both of which were abrogated by additional TREM2 knockout. Knocking out TREM2 in 5xFAD mice exacerbated Aß pathology and neurodegeneration but reduced Iba1+ cell numbers, all of which could not be rescued by additional CD33 knockout. RNA-seq profiling of microglia revealed that genes related to phagocytosis and signaling (IL-6, IL-8, acute phase response) are upregulated in 5xFAD;CD33-/- and downregulated in 5xFAD;TREM2-/- mice. Differential gene expression in 5xFAD;CD33-/- microglia depended on the presence of TREM2, suggesting TREM2 acts downstream of CD33. Crosstalk between CD33 and TREM2 includes regulation of the IL-1ß/IL-1RN axis and a gene set in the "receptor activity chemokine" cluster. Our results should facilitate AD therapeutics targeting these receptors.