Axon pathology in mouse models of Huntington's disease

Axon or synapse dysfunction parallels or precedes symptom onset in many neurodegenerative disorders. In some of these conditions, not only do axon and synapse loss determine the course of pathology, but protection of those neuronal compartments is mandatory to alleviate the disease. Whether this is also the case in Huntington’s disease (HD), a devastating neurodegenerative disorder characterised by progressive deterioration of both physical and mental abilities and inevitable early death, remains unclear. Present therapeutic strategies do not address protection of axons and synapses, which may help explain why there is no effective treatment currently in use. Moreover, an accurate characterisation of the development of axon pathology relative to neuronal loss and to the deposition of mutant-Huntingtin (mHTT) aggregates, neuropathological hallmark of HD, is lacking. In the present thesis I have carried out a detailed study aiming to investigate axon degeneration in the R6/2 transgenic (Tg) and the HdhQ140 knock-in (KI) mice, two HD models, and to assess whether this occurs early in and contributes to the course of pathology. I tested the hypothesis that axon degeneration precedes or at least parallels degeneration of other neuronal compartments in these mice. To characterise axon pathology and its spatio-temporal relationship to aggregate formation, neuronal loss and symptom onset, I crossed R6/2 and HdhQ140 mice with YFP-H transgenic mice that express the yellow fluorescent protein (YFP) in a subset of neurons. Neuronal pathways labelled by YFP in this model include some reported to be affected in HD. In these mice individual fluorescent neurons can be tracked over long distance and axons can be traced back to their cell bodies. Using a powerful axon imaging method that was developed and successfully applied to study Alzheimer’s disease, I was able to place axon degeneration accurately in the sequence of pathological events and develop methods to quantify it as readout for future therapeutic studies from our group and elsewhere. I found that the morphology of axons was strikingly abnormal in some brain areas in HdhQ140 homozygous mice (HdhQ140/Q140) where large axonal swellings were detected at 6 months and at 12 months of age in stria terminalis and striatum. In these mice, the number of axonal swellings increased age-dependently and was significantly higher than that found in wild-type littermates. However, I did not detect degeneration in cell bodies, dendrites or synapses suggesting that axon pathology is the main feature of the disease in this model. To better characterise the KI model, I also performed a battery of behavioural tests to assess motor and cognitive impairment during disease progression. I used tests of locomotor activity, motor coordination and balance and sensorimotor gating to measure motor function and tests of spatial working memory and anxiety-like behaviour to assess cognitive and behavioural symptoms, respectively. A longitudinal study from 1 to 12 months was carried out to detect pathological changes from early stage and relate them to swelling formation. In all tests, I found a strong reduction in locomotor activity in HdhQ140 mice compared to the controls although balance and coordination seemed not to be impaired as rotarod performance was unaltered. Alterations were also detected in prepulse inhibition, suggesting sensorimotor defects occur in these mice, while no abnormal cognitive or psychiatric behaviour was detected in the time-frame of the study. Behavioural symptoms, as well as abnormal morphological changes found in axons, worsened with time and major impairments were found at the latest time-point, 12 months of age. Finally, I asked whether alterations in the NAD biosynthetic pathway could underline the signs of axon pathology detected in HdhQ140 homozygous mice, as it has recently emerged that this pathway regulates axon survival and axon and synapse degeneration in many neurodegenerative disorders. To this purpose, I looked at possible alterations in the level of nucleotides (NMN, NAD) and in the activity of key enzymes in this pathway (NMNAT, NAMPT). I also tested the hypothesis that mHTT interacts with NMNAT enzymes and with the Wallerian degeneration slow protein (WLDs), an NMNAT fusion protein, and interferes/impairs their normal function. As WLDs delays axon degeneration in acute and neurodegeneration models, future works may address beneficial role of WLDs in HD/WldS crossed mice. Despite no detected alterations in nucleotide levels or enzymatic activity in the KI mice compared to the controls, colocalisation was found between mHTT and WLDs and between mHTT and NMNAT2, an important axon survival factor, suggesting a possible interaction between these proteins which could play a role in HD neurodegeneration

Axon pathology in mouse models of Huntington's disease

Axon or synapse dysfunction parallels or precedes symptom onset in many neurodegenerative disorders. In some of these conditions, not only do axon and synapse loss determine the course of pathology, but protection of those neuronal compartments is mandatory to alleviate the disease. Whether this is also the case in Huntington’s disease (HD), a devastating neurodegenerative disorder characterised by progressive deterioration of both physical and mental abilities and inevitable early death, remains unclear. Present therapeutic strategies do not address protection of axons and synapses, which may help explain why there is no effective treatment currently in use. Moreover, an accurate characterisation of the development of axon pathology relative to neuronal loss and to the deposition of mutant-Huntingtin (mHTT) aggregates, neuropathological hallmark of HD, is lacking. In the present thesis I have carried out a detailed study aiming to investigate axon degeneration in the R6/2 transgenic (Tg) and the HdhQ140 knock-in (KI) mice, two HD models, and to assess whether this occurs early in and contributes to the course of pathology. I tested the hypothesis that axon degeneration precedes or at least parallels degeneration of other neuronal compartments in these mice. To characterise axon pathology and its spatio-temporal relationship to aggregate formation, neuronal loss and symptom onset, I crossed R6/2 and HdhQ140 mice with YFP-H transgenic mice that express the yellow fluorescent protein (YFP) in a subset of neurons. Neuronal pathways labelled by YFP in this model include some reported to be affected in HD. In these mice individual fluorescent neurons can be tracked over long distance and axons can be traced back to their cell bodies. Using a powerful axon imaging method that was developed and successfully applied to study Alzheimer’s disease, I was able to place axon degeneration accurately in the sequence of pathological events and develop methods to quantify it as readout for future therapeutic studies from our group and elsewhere. I found that the morphology of axons was strikingly abnormal in some brain areas in HdhQ140 homozygous mice (HdhQ140/Q140) where large axonal swellings were detected at 6 months and at 12 months of age in stria terminalis and striatum. In these mice, the number of axonal swellings increased age-dependently and was significantly higher than that found in wild-type littermates. However, I did not detect degeneration in cell bodies, dendrites or synapses suggesting that axon pathology is the main feature of the disease in this model. To better characterise the KI model, I also performed a battery of behavioural tests to assess motor and cognitive impairment during disease progression. I used tests of locomotor activity, motor coordination and balance and sensorimotor gating to measure motor function and tests of spatial working memory and anxiety-like behaviour to assess cognitive and behavioural symptoms, respectively. A longitudinal study from 1 to 12 months was carried out to detect pathological changes from early stage and relate them to swelling formation. In all tests, I found a strong reduction in locomotor activity in HdhQ140 mice compared to the controls although balance and coordination seemed not to be impaired as rotarod performance was unaltered. Alterations were also detected in prepulse inhibition, suggesting sensorimotor defects occur in these mice, while no abnormal cognitive or psychiatric behaviour was detected in the time-frame of the study. Behavioural symptoms, as well as abnormal morphological changes found in axons, worsened with time and major impairments were found at the latest time-point, 12 months of age. Finally, I asked whether alterations in the NAD biosynthetic pathway could underline the signs of axon pathology detected in HdhQ140 homozygous mice, as it has recently emerged that this pathway regulates axon survival and axon and synapse degeneration in many neurodegenerative disorders. To this purpose, I looked at possible alterations in the level of nucleotides (NMN, NAD) and in the activity of key enzymes in this pathway (NMNAT, NAMPT). I also tested the hypothesis that mHTT interacts with NMNAT enzymes and with the Wallerian degeneration slow protein (WLDs), an NMNAT fusion protein, and interferes/impairs their normal function. As WLDs delays axon degeneration in acute and neurodegeneration models, future works may address beneficial role of WLDs in HD/WldS crossed mice. Despite no detected alterations in nucleotide levels or enzymatic activity in the KI mice compared to the controls, colocalisation was found between mHTT and WLDs and between mHTT and NMNAT2, an important axon survival factor, suggesting a possible interaction between these proteins which could play a role in HD neurodegeneration

AMPK in the central nervous system: physiological roles and pathological implications

5′ AMP-activated protein kinase (AMPK) is considered the master metabolic regulator in all eukaryotes, as it maintains cellular energy homeostasis in a variety of tissues, including the brain. In humans, alterations in AMPK activity can lead to a wide spectrum of metabolic disorders. The relevance of this protein kinase in the pathogenesis of diabetes and metabolic syndrome is now well established. On the contrary, correlations between AMPK and brain physiopathology are still poorly characterized. The aim of this review is to summarize and discuss the current knowledge about the prospective involvement of AMPK in the onset and the progression of different neurological diseases

Age-related axonal swellings precede other neuropathological hallmarks in a knock-in mouse model of Huntington's disease.

Axon degeneration precedes cell body death in many age-related neurodegenerative disorders, often determining symptom onset and progression. A sensitive method for revealing axon pathology could indicate whether this is the case also in Huntington's disease (HD), a fatal, devastating neurodegenerative disorder causing progressive deterioration of both physical and mental abilities, and which brain region is affected first. We studied the spatio-temporal relationship between axon pathology, neuronal loss, and mutant Huntingtin aggregate formation in HD mouse models by crossing R6/2 transgenic and HdhQ140 knock-in mice with YFP-H mice expressing the yellow fluorescent protein in a subset of neurons. We found large axonal swellings developing age-dependently first in stria terminalis and then in corticostriatal axons of HdhQ140 mice, whereas alterations of other neuronal compartments could not be detected. Although mutant Huntingtin accumulated with age in several brain areas, inclusions in the soma did not correlate with swelling of the corresponding axons. Axon abnormalities were not a prominent feature of the rapid progressive pathology of R6/2 mice. Our findings in mice genetically similar to HD patients suggest that axon pathology is an early event in HD and indicate the importance of further studies of stria terminalis axons in man

GATA3 and MDM2 are synthetic lethal in estrogen receptor-positive breast cancers.

Synthetic lethal interactions, where the simultaneous but not individual inactivation of two genes is lethal to the cell, have been successfully exploited to treat cancer. GATA3 is frequently mutated in estrogen receptor (ER)-positive breast cancers and its deficiency defines a subset of patients with poor response to hormonal therapy and poor prognosis. However, GATA3 is not yet targetable. Here we show that GATA3 and MDM2 are synthetically lethal in ER-positive breast cancer. Depletion and pharmacological inhibition of MDM2 significantly impaired tumor growth in GATA3-deficient models in vitro, in vivo and in patient-derived organoids/xenograft (PDOs/PDX) harboring GATA3 somatic mutations. The synthetic lethality requires p53 and acts via the PI3K/Akt/mTOR pathway. Our results present MDM2 as a therapeutic target in the substantial cohort of ER-positive, GATA3-mutant breast cancer patients. With MDM2 inhibitors widely available, our findings can be rapidly translated into clinical trials to evaluate in-patient efficacy

Association of kidney disease measures with risk of renal function worsening in patients with type 1 diabetes

Background: Albuminuria has been classically considered a marker of kidney damage progression in diabetic patients and it is routinely assessed to monitor kidney function. However, the role of a mild GFR reduction on the development of stage 653 CKD has been less explored in type 1 diabetes mellitus (T1DM) patients. Aim of the present study was to evaluate the prognostic role of kidney disease measures, namely albuminuria and reduced GFR, on the development of stage 653 CKD in a large cohort of patients affected by T1DM. Methods: A total of 4284 patients affected by T1DM followed-up at 76 diabetes centers participating to the Italian Association of Clinical Diabetologists (Associazione Medici Diabetologi, AMD) initiative constitutes the study population. Urinary albumin excretion (ACR) and estimated GFR (eGFR) were retrieved and analyzed. The incidence of stage 653 CKD (eGFR < 60 mL/min/1.73 m2) or eGFR reduction > 30% from baseline was evaluated. Results: The mean estimated GFR was 98 \ub1 17 mL/min/1.73m2 and the proportion of patients with albuminuria was 15.3% (n = 654) at baseline. About 8% (n = 337) of patients developed one of the two renal endpoints during the 4-year follow-up period. Age, albuminuria (micro or macro) and baseline eGFR < 90 ml/min/m2 were independent risk factors for stage 653 CKD and renal function worsening. When compared to patients with eGFR > 90 ml/min/1.73m2 and normoalbuminuria, those with albuminuria at baseline had a 1.69 greater risk of reaching stage 3 CKD, while patients with mild eGFR reduction (i.e. eGFR between 90 and 60 mL/min/1.73 m2) show a 3.81 greater risk that rose to 8.24 for those patients with albuminuria and mild eGFR reduction at baseline. Conclusions: Albuminuria and eGFR reduction represent independent risk factors for incident stage 653 CKD in T1DM patients. The simultaneous occurrence of reduced eGFR and albuminuria have a synergistic effect on renal function worsening

Implementation of clinical exome sequencing in prenatal setting: comparing between prospective and retrospective cohort studies

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en Sciences biomédicales et pharmaceutiques (Médecine)info:eu-repo/semantics/nonPublishe